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|DR.  WILLIAM  J.  GIES  J? 

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ofthe 

G1ES  FELLOWSHIP 

in  Biological  Chemistry 


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http://www.archive.org/details/practicalphysiolOOhawk 


PRACTICAL 
PHYSIOLOGICAL    CHEMISTRY 


HAWK 


Absorption  Spectra. 


PLATE    I. 


3     <& 


Oxyhaemoglobln. 


Haemoglobin. 


Carboxy- 
haemoglobin. 


Neutral  Met- 

haemoglobin. 


Alkaline  Met- 
haemoglobin. 


Alkali 
Haematin. 


Absorption  Spectra. 


PLATE      H. 


Reduced  Alkali 
Haematin  or 
Haemochromogen. 


Acid  Haematin  in 
ethereal  solution. 


Acid  Haemato- 
porphyrin. 


Alkaline 
Haematopor- 

phyrin. 


Urobilin  or  Hydro- 
bilirubin  in  acid 
solution. 


Urobilin  or  Hydro- 
bilirubin  in  alkaline 
solution  after  the 
addition  of  zinc 
chloride  solution. 


Bilicyanin  or 
Cholecyanin  in 
alkaline  solution. 


0 
PRACTICAL 


PHYSIOLOGICAL    CHEMISTRY 


A  BOOK  DESIGNED  FOR  USE  IN  COURSES 
IN  PRACTICAL  PHYSIOLOGICAL  CHEMISTRY 
IN  SCHOOLS  OF  MEDICINE  AND  OF  SCIENCE 


BY 

PHILIP  B.  HAWK,  M.S.,  Ph.D. 

PROFESSOR    OF    PHYSIOLOGICAL   CHEMISTRY    IN   THE   UNIVERSITY    OF    ILLINOIS 


WITH  TWO  FULL  PAGE  PLATES  OF  ABSORPTION  SPECTRA  IN  COLORS, 
FOUR  ADDITIONAL  FULL  PAGE  COLOR  PLATES  AND  ONE  HUN- 
DRED AND    TWENTY-SIX  FIGURES   OF   WHICH 
TWELVE  ARE  FN  COLORS 


SECOND  EDITION,  REVISED  AND  ENLARGED 


PHILADELPHIA 

P.  BLAKISTON'S  SON  &  CO. 

10I2    WALNUT    STREET 
I9O9 


PREFACE  TO  SECOND  EDITION 

The  kind  reception  accorded  this  volume  by  the  instructors  in 
physiological  chemistry  in  the  United  States  and  Great  Britain  has 
made  the  preparation  of  a  new  edition  imperative,  notwithstanding 
the  fact  that  less  than  two  years  have  elapsed  since  the  former 
edition  appeared.  The  advance  and  development  made  in  the  field 
of  physiological  chemistry  during  this  period  have  been  both  rapid 
and  important;  conditions  which  would  of  themselves  have  neces- 
sitated the  revision  of  the  volume  at  an  early  date. 

The  book  has  been  thoroughly  revised  in  all  departments  and  in 
part  rewritten,  the  system  of  spelling  officially  adopted  by  the 
American  Chemical  Society  having  been  followed  throughout  the 
volume.  Besides  introducing  many  neAv  qualitative  tests  and  quan- 
titative methods,  the  author  has  added  a  chapter  on  "  Enzymes  and 
Their  Action  "  and  has  rewritten  the  two  chapters  on  Proteins. 
The  term  "  protein "  has  been  substituted  for  "  proteid "  and  the 
classification  of  proteins  as  recently  adopted  by  the  American  Physi- 
ological Society  and  the  American  Society  of  Biological  Chemists 
has  been  introduced  and  is  followed  throughout  the  text ;  the  classi- 
fication adopted  by  the  British  Medical  Association  is  also  included. 

The  original  plan  of  the  book  has  been  adhered  to  with  the  excep- 
tion that  the  chapter  on  "  Enzymes  and  Their  Action  "  has  been 
made  Chapter  I  and  the  practical  work  upon  the  proteins  is  pre- 
ceded by  a  chapter  giving  a  brief  discussion  of  protein  substances 
from  the  standpoint  of  their  decomposition  and  synthesis.  We 
believe  that  the  student  will  be  able  to  pursue  his  practical  work 
more  intelligently  and  will  derive  greater  benefit  therefrom  if  the 
plan  of  instruction  as  suggested  in  Chapters  IV  and  V  be  followed 
in  the  presentation  of  the  subject  of  "  Proteins." 

The  author  wishes  to  express  his  thanks  to  all  those  who  so  kindly 
offered  suggestions  for  the  betterment  of  the  book.  He  is  particu- 
larly desirous  of  expressing  his  gratitude  to  Professor  Lafayette  B. 
Mendel  and  Dr.  Thomas  B.  Osborne  for  the  many  helpful  sugges- 
tions they  have  so  kindly  given  him.  His  thanks  are  also  due  Pro- 
fessor C.  A.  Herter,  Dr.  H.  D.  Dakin,  Dr.  S.  R.  Benedict  and  Mr. 
S.  C.  Clark  for  permission  to  insert  unpublished  material,  to  Mr. 
Paul  E.  Howe  for  valuable  assistance  rendered  in  the  reading  of 


Vlll  PREFACE    TO    SECOND    EDITION. 

proof  and  in  the  verification  of  tests  and  methods,  and  to  Dr.  M.  E. 
Rehfuss  for  assistance  in  proof  reading. 

The  author  takes  this  opportunity  of  making  an  acknowledg- 
ment which  was  inadvertently  omitted  from  the  first  edition.  He 
wishes  to  express  his  obligation  to  the  laboratories  of  physiological 
chemistry  at  Yale  University  and  at  Columbia  University  (College 
of  Physicians  and  Surgeons)  in  the  latter  of  which  he  was  Assistant 
to  Professor  W.  J.  Gies  for  two  years.  The  courses  given  in  these 
laboratories  formed  the  basis  of  many  of  the  experiments  included 
in  this  volume  and  it  is  with  feelings  of  deepest  gratitude  that  he 
records  this  acknowledgment  of  the  assistance  thus  rendered  by 
those  in  charge  of  these  courses. 

Philip  B.  Hawk. 

Urbana,  Illinois, 
Fehruary  I,  1909. 


PREFACE  TO  FIRST  EDITION 

The  plan  followed  in  the  presentation  of  the  subject  of  this 
volume  is  rather  different,  so  far  as  the  author  is  aware,  from  that 
set  forth  in  any  similar  volume.  This  plan,  however,  he  feels  to 
be  a  logical  one  and  has  followed  it  with  satisfactory  results  during 
a  period  of  three  years  in  his  own  classes  at  the  University  of  Penn- 
sylvania. The  main  point  in  which  the  plan  of  the  author  differs 
from  those  previously  proposed  is  in  the  treatment  of  the  food  stuffs 
and  their  digestion. 

In  Chapter  IV  the  "  Decomposition  Products  of  Proteids  "  has 
been  treated  although  it  is  impracticable  to  include  the  study  of  this 
topic  in  the  ordinary  course  in  practical  physiological  chemistry. 
For  the  specimens  of  the  decomposition  products,  the  crystalline 
forms  of  which  are  reproduced  by  original  drawings  or  by  micro- 
photographs,  the  author  is  indebted  to  Dr.  Thomas  B.  Osborne,  of 
New  Haven,  Conn. 

Because  of  the  increasing  importance  attached  to  the  examina- 
tion of  feces  for  purposes  of  diagnosis,  the  author  has  devoted  a 
chapter  to  this  subject.  He  feels  that  a  careful  study  of  this  topic 
deserves  to  be  included  in  the  courses  in  practical  physiological 
chemistry,  of  medical  schools  in  particular.  The  subject  of  solid 
tissues  (Chapters  XIII,  XIV  and  XV)  has  also  been  somewhat 
more  fully  treated  than  has  generally  been  customary  in  books  of 
this  character. 

The  author  is  deeply  indebted  to  Professor  Lafayette  B.  Mendel, 
of  Yale  University,  for  his  careful  criticism  of  the  manuscript  and 
to  Professor  John  Marshall,  of  the  University  of  Pennsylvania,  for 
his  painstaking  revision  of  the  proof.  He  also  wishes  to  express 
his  gratitude  to  Dr.  David  L.  Edsall  for  his  criticism  of  the  clinical 
portion  of  the  volume ;  to  Dr.  Otto  Folin  for  suggestions  regarding 
several  of  his  quantitative  methods,  and  to  Mr.  John  T.  Thomson 
for  assistance  in  proof  reading. 

For  the  micro-photographs  of  oxyhemoglobin  and  haemin  repro- 
duced in  Chapter  XI  the  author  is  indebted  to  Professor  E.  T. 
Reichert,  of  the  University  of  Pennsylvania,  who,  in  collaboration 
with  Professor  A.  P.  Brown,  of  the  University  of  Pennsylvania,  is 
making  a  very  extended  investigation  into  the  crystalline  forms  of 


X  PREFACE    TO    FIRST    EDITION. 

biochemic  substances.  The  micro-photograph  of  allantoin  was 
kindly  furnished  by  Professor  Mendel.  The  author  is  also  indebted 
for  suggestions  and  assistance  received  from  the  lectures  and  pub- 
lished writings  of  numerous  authors  and  investigators. 

The  original  drawings  of  the  volume  were  made  by  Mr.  Louis 
Schmidt  whose  eminently  satisfactory  efforts  are  highly  appreciated 
by  the  author. 

Philip  B.  Hawk. 

Philadelphia. 


CONTENTS 


CHAPTER  I. 
Enzymes  and  Their  Action i 

CHAPTER  II. 
Carbohydrates 21 

CHAPTER  III. 
Salivary  Digestion    53 

CHAPTER  IV. 
Proteins:  Their  Decomposition  and  Synthesis 61 

CHAPTER  V. 
Proteins  :  Their  Classification  and  Properties 85 

CHAPTER  VI. 
Gastric  Digestion    118 

CHAPTER  VII. 
Fats 131 

CHAPTER  VIII. 
Pancreatic  Digestion 140 

CHAPTER  IX. 
Bile    150 

CHAPTER  X. 
Putrefaction  Products   162 

CHAPTER  XL 
Feces   172 

CHAPTER  XII. 
Blood  182 

xi 


Xll  CONTENTS. 

CHAPTER  XIII. 
Milk 218 

CHAPTER  XIV. 
Epithelial  and  Connective  Tissues 227 

CHAPTER  XV. 
Muscular  Tissue   235 

CHAPTER  XVI. 
Nervous  Tissue 248 

CHAPTER  XVII. 
Urine  :  General  Characteristics  of  Normal  and  Path- 
ological Urine   254 

CHAPTER  XVIII. 
Urine  :  Physiological  Constituents 264 

CHAPTER  XIX. 
Urine  :  Pathological  Constituents 305 

CHAPTER  XX. 
Urine:  Organized  and  Unorganized  Sediments  .  . 343 

CHAPTER  XXI. 
Urine  :  Calculi 362 

CHAPTER  XXII. 
Urine  :  Quantitative  Analysis 366 

CHAPTER  XXIII. 
Quantitative    Analysis    of    Milk,    Gastric    Juice    and 

Blood  410 

Index 427 


LIST  OF  ILLUSTRATIONS 


Plate 

I.  Absorption  Spectral  „       ..  L. 

TT     . ,  .       _r  \- Frontispiece 

11.  Absorption  Spectra  J 

III.  Osazones Opposite  page  24 

IV.  Normal  Erythrocytes  and  Leucocytes Opposite  page  184 

V.  Uric  Acid  Crystals Opposite  page  273 

VI.  Ammonium  Urate   Opposite  page  348 

Figure  Page 

1.  Dialyzing  Apparatus  for  Students'  Use 25 

2.  Einhorn  Saccharometer 31 

3.  One  Form  of  Laurent  Polariscope 33 

4.  Diagrammatic  Representation  of  the  course  of  the  Light 

through  the  Laurent  Polariscope 33 

5.  Polariscope  (Schmidt  and  Hansen  Model) 34 

6.  Iodoform   42 

7.  Potato  Starch 45 

8.  Bean  Starch 45 

9.  Arrowroot  Starch 45 

10.  Rye  Starch 45 

11.  Barley  Starch 45 

12.  Oat  Starch    45 

13.  Buckwheat  Starch 45 

14.  Maize  Starch 45 

15.  Rice  Starch 45 

16.  Pea  Starch 45 

17.  Wheat  Starch   45 

18.  Microscopical  Constituents  of  Saliva 56 

19.  Glycocoll  Ester  Hydrochloride   68 

20.  Serine 69 

21.  Phenylalanine    70 

22.  Fischer  Apparatus    71 

23.  Tyrosine J2 

24.  Cystine y^ 

25.  Histidine  Dichloride 74 

26.  Leucine    76 

xiii 


XIV  LIST    OF    ILLUSTRATIONS. 

Figure  Page 

27.  Lysine  Picrate 78 

28.  Aspartic  Acid   78 

29.  Glutamic  Acid 79 

30.  Levo-a-Proline 80 

31.  Copper  Salt  of  Proline  .  .  .  .  . 81 

32.  Coagulation  Temperature  Apparatus   100 

33.  Edestin    104 

34.  Excelsin,  the  Protein  of  the  Brazil  Nut 105 

35.  Beef  Fat 131 

36.  Mutton  Fat '. 134 

37.  Pork  Fat   136 

38.  Palmitic  Acid    137 

39.  Melting-Point  Apparatus 138 

40.  Bile  Salts 152 

41.  Bilirubin   (Hematoidin)    153 

42.  Cholesterol 159 

43.  Taurine    160 

44.  Glycocoll 161 

45.  Ammonium  Chloride    166 

46.  Microscopical  Constituents  of  Feces 172 

47.  Hematoidin  Crystals  from  Acholic  Stools 173 

48.  Charcot-Leyden  Crystals 174 

49.  Boas'  Sieve   177 

50.  Oxyhemoglobin  Crystals  from  Blood  of  the  Guinea  Pig.  186 

51.  Oxyhemoglobin  Crystals  from  Blood  of  the  Rat 186 

52.  Oxyhemoglobin  Crystals  from  Blood  of  the  Horse 187 

53.  Oxyhemoglobin  Crystals  from  Blood  of  the  Squirrel  .  .  .  187 

54.  Oxyhemoglobin  Crystals  from  Blood  of  the  Dog 188 

55.  Oxyhemoglobin  Crystals  from  Blood  of  the  Cat 188 

56.  Oxyhemoglobin  Crystals  from  Blood  of  the  Necturus  .  .  189 

57.  Effect  of  Water  on  Erythrocytes 195 

58.  Hemin  Crystals  from  Human  Blood 198 

59.  Hemin  Crystals  from  Sheep  Blood , 198 

60.  Sodium  Chloride ' 200 

61.  Direct -vision  Spectroscope 203 

62.  Angular-vision    Spectroscope   Arranged    for   Absorption 

Analysis 204 

63.  Diagram  of  Angular-vision  Spectroscope   204 

64.  Fleischl's  TIemometer   208 

65.  Pipette  of  Fleischl's  Hemometer 208 


LIST    OF    ILLUSTRATIONS.  XV 

Figure  Page 

66.  Colored  Glass  Wedge  of  Fleischl's  Hiemometer 209 

6y.  Dare's  Hsemoglobinometer 210 

68.  Horizontal  Section  of  Dare's  Hsemoglobinometer 211 

69.  Method   of    Filling   the    Capillary   Observation    Cell    of 

Dare's  Hasmoglobinometer   212 

70.  Thoma-Zeiss  Counting  Chamber    213 

71.  Thoma-Zeiss  Capillary  .Pipettes    214 

72.  Ordinary  Ruling  of  Thoma-Zeiss  Counting  Chamber  ...  215 

73.  Zappert's    Modified    Ruling    of    Thoma-Zeiss    Counting 

Chamber    216 

74.  Normal  Milk  and  Colostrum 219 

75.  Lactose 220 

76.  Calcium  Phosphate 224 

yy.  Creatine    238 

78.  Xanthine    239 

79.  Hypoxanthine  Silver  Nitrate 245 

80.  Xanthine  Silver  Nitrate 247 

81.  Deposit  in  Ammoniacal  Fermentation   257 

82.  Deposit  in  Acid  Fermentation 257 

83.  Urinometer  and  Cylinder 258 

84.  Beckmann-Heidenhain  Freezing-Point  Apparatus 260 

85.  Urea 266 

86.  Urea  Nitrate 268 

87.  Melting-Point  Tubes  Fastened  to  Bulb  of  Thermometer.  269 

88.  Urea  Oxalate    .  .  . 270 

89.  Pure  Uric  Acid 274 

90.  Creatinine    277 

91.  Creatinine-Zinc  Chloride   278 

92.  Hippuric  Acid 282 

93.  Allantoin  from  Cat's  Urine 286 

94.  Benzoic  Acid    289 

95.  Calcium  Sulphate   298 

96.  "  Triple  Phosphate  "    .' 301 

97.  The  Purdy  Electric  Centrifuge 343 

98.  Sediment  Tube  for  the  Purdy  Electric  Centrifuge 343 

99.  Calcium  Oxalate 345 

100.  Calcium    Carbonate    346 

101.  Various  Forms  of  Uric  Acid 347 

102.  Acid  Sodium  Urate   348 

103.  Cystine   349 


XVI  LIST    OF    ILLUSTRATIONS. 

Figure  Page 

104.  Crystals  of  Impure  Leucine 350 

105.  Epithelium  from  Different  Areas  of  the  Urinary  Tract.  .  352 

106.  Pus  Corpuscles   353 

107.  Hyaline  Casts   354 

108.  Granular  Casts 355 

109.  Granular  Casts   356 

1 10.  Epithelial   Casts    ■ 356 

in.   Blood,  Pus,  Hyaline  and  Epithelial  Casts   356 

1 12.  Fatty  Casts   357 

113.  Fatty  and  Waxy  Casts 357 

1 14.  Cylindroids    ' 358 

115.  Crenated  Erythrocytes 359 

116.  Human  Spermatozoa   360 

117.  Esbach's  Albuminometer 367 

118.  Marshall's  Urea  Apparatus 375 

1 19.  Hiifner's  Urea  Apparatus 377 

120.  Doremus-Hinds  Ureometer 378 

121.  Folin's  Urea  Apparatus 379 

122.  Folin's  Ammonia  Apparatus 380 

123.  Folin  Absorption  Tube 381 

124.  Berthelot-Atwater  Bomb  Calorimeter    388 

125.  Soxhlet  Apparatus   410 

126.  Feser's  Lactoscope 41 1 


PHYSIOLOGICAL   CHEMISTRY. 


CHAPTER  I. 

ENZYMES  AND  THEIR  ACTION. 

According  to  the  old  classification  ferments  were  divided  into 
two  classes,  the  organized  ferments  and  the  unorganized  ferments. 
As  organized  ferments  or  true  ferments  there  were  grouped  such 
substances  as  yeast  and  certain  bacteria  which  were  supposed  to 
act  by  virtue  of  vital  processes,  whereas  the  unorganized  ferments 
included  salivary  amylase  (ptyalin),  gastric  protease  (pepsin), 
pancreatic  protease  (trypsin),  etc.,  which  were  described  as  "non- 
living unorganized  substances  of  a  chemical  nature."  Kuhne  des- 
ignated this  latter  class  of  substances  as  enzymes  (iv  typy — in 
yeast).  This  division  into  organized  ferments  (true  ferments)  and 
unorganized  ferments  (enzymes)  was  generally  accepted  and  was 
practically  unquestioned  until  Buchner  overthrew  it  in  the  year 
1897  by  his  epoch-making  investigations  on  zymase.  Previous  to 
this  time  many  writers  had  expressed  the  opinion  that  the  action  of 
the  ferment  organisms  was  similar  to  that  of  the  unorganized 
ferments  or  enzymes  and  that  therefore  the  activity  of  the  former 
was  possibly  due  to  the  production  of  a  substance  in  the  cell,  which 
was  in  nature  similar  to  an  enzyme.  Investigation  after  investiga- 
tion, however,  failed  to  isolate  any  such  principle  from  an  active 
cell  and  the  exponents  of  the  "  vital  "  theory  became  strengthened 
in  their  belief  that  certain  fermentative  processes  brought  about 
by  living  cells  could  not  occur  apart  from  the  biological  activity 
of  such  cells.  However,  as  early  as  1858,  Traube  had  enunciated, 
in  substance,  the  principles  which  were  destined  to  be  fundamental 
in  our  modern  theory  of  fermentation.  He  expressed  the  belief 
that  the  yeast  cell  produced  a  product  in  its  metabolic  activities 
which  had  the  property  of  reacting  with  sugar  with  the  production 
of  carbon  dioxide  and  alcohol,  and  further  that  this  reaction  be- 
tween the  product  of  the  metabolism  of  the  yeast  cell  and  the  sugar 
2  1 


2  PHYSIOLOGICAL    CHEMISTRY. 

occurred  without  aid  from  the  original  cell.  It  was  not  until  1897, 
however,  that  this  theory  was  placed  upon  a  firm  experimental 
basis.  This  was  brought  about  through  the  efforts  of  Buchner 
who  succeeded  in  isolating  from  the  living  yeast  cells  a  substance 
(zymase)  which,  when  freed  from  the  last  trace  of  organized  cel- 
lular material,  was  able  to  bring  about  the  identical  fermentative 
processes,  which,  up  to  this  time,  had  been  deemed  possible  only 
in  the  presence  of  the  active,  living  yeast  cell. 

Buchner's  manipulation  of  the  yeast  cells  consisted  in  first  grind- 
ing them  with  sand  and  infusorial  earth  after  which  the  finely 
divided  material  was  subjected  to  great  pressure  (300  atmospheres) 
and  yielded  a  liquid  which  possessed  the  fermentative  activity  of 
the  unchanged  yeast  cell.1  This  liquid  contained  zymase,  the  prin- 
cipal enzyme  of  the  yeast  cell.  Later  the  lactic-acid-  and  acetic- 
acid-producing  bacteria  were  subjected  by  Buchner  to  treatment 
similar  to  that  accorded  the  yeast  cells,  and  the  active  intracellular 
enzymes  were  obtained.  Many  other  instances  are  on  record  in 
which  a  soluble,  active,  agent  has  been  isolated  from  ferment 
cells,  with  the  result  that  it  is  pretty  well  established  that  all  the 
so-called  organized  ferments  elaborate  substances  of  this  character. 
Therefore,  basing  our  definition  on  the  work  of  Buchner  and 
others  we  may  define  an  enzyme,  as  an  unorganised,  soluble  ferment, 
which  is  elaborated  by  an  animal  or  vegetable  cell  and  wihose  ac- 
tivity is  entirely  independent  of  any  of  the  life  processes  of  such 
a  cell.  According  to  this  definition  the  enzyme  zymase  elaborated 
by  the  yeast  cell  is  entirely  comparable  to  the  enzyme  pepsin  elabor- 
ated by  the  cells  of  the  stomach  mucosa.  One  is  derived  from  a 
vegetable  cell,  the  other  from  an  animal  cell,  yet  the  activity  of 
neither  is  dependent  upon  the  integrity  of  the  cell. 

Enzymes  act  by  catalysis  and  hence  may  be  termed  catalyzers 
or  catalysts.  A  simple  rough  definition  of  a  catalyzer  is  "  a  sub- 
stance which  alters  the  velocity  of  a  chemical  reaction  without  un- 
dergoing any  apparent  physical  or  chemical  change  itself  and  with- 
out becoming  a  part  of  the  product  formed."  It  is  a  well-known  fact 
that  the  velocity  of  the  greater  number  of  chemical  reactions  may 
be  changed  through  the  presence  of  some  catalyzer.  For  example, 
take  the  case  of  hydrogen  peroxide.  It  spontaneously  decomposes 
slowly  into  water  and  oxygen.     In  the  presence  of  colloidal  plati- 

1  In  later  investigations  the  process  was  improved  by  freezing  the  ground  cells 
with  liquid  air  and  finely  pulverizing  them,  before  applying  the  pressure. 


ENZYMES    AND    THEIR    ACTION.  3 

num,1  however,  the  decomposition  is  much  accelerated  and  ceases 
only  when  the  destruction  of  the  hydrogen  peroxide  is  complete. 
Without  multiplying  instances,  suffice  it  to  say  that  there  is  an 
analogy  between  inorganic  catalyzers  and  enzymes,  the  main  point 
of  difference  between  the  enzymes  and  most  of  the  inorganic  cataly- 
zers being  that  the  enzymes  are  colloids.2 

Inasmuch  as  each  of  the  enzymes  has  an  action  which  is  more 
or  less  specific  in  character,  and  since  it  is  a  fairly  simple  matter, 
ordinarily,  to  determine  the  character  of  that  action,  the  classifi- 
cation of  the  enzymes  is  not  attended  with  very  great  difficulties. 
They  are  ordinarily  classified  according  to  the  nature  of  the  sub- 
strate3 or  according  to  the  type  of  reaction  they  bring  about.  Thus 
we  have  various  classes  of  enzymes  such  as  amylolytic*  proteolytic, 
lipolytic,  glycolytic,  mucolytic,  autolytic,  oxidizing,  reducing,  in- 
verting, protein-coagulating,  deaniidizing,  etc.  In  every  instance 
the  class  name  indicates  the  individual  type  of  enzymatic  activity 
which  the  enzymes  included  in  that  class  are  capable  of  accomplish- 
ing. For  example,  amylolytic  enzymes  facilitate  the  hydrolysis  of 
starch  (amylum)  and  related  substances,  lipolytic  enzymes  facilitate 
the  hydrolysis  of  fats  (Xi7ro?)  whereas  through  the  agency  of  uri- 
colytic enzymes,  uric  acid  is  broken  down.  There  is  a  tendency,  at 
the  present  time,  to  harmonize  the  nomenclature  of  the  enzymes 
by  the  use  of  the  termination,  -ase.  According  to  this  system  of 
nomenclature,  all  starch-transforming  enzymes,  or  so-called  amy- 
lolytic enzymes,  are  called  amylases,  all  fat-splitting  enzymes  are 
called  lipases,  etc.  Thus  ptyalin  the  amylolytic  enzyme  of  the 
saliva,  would  be  termed  salivary  amylase  in  order  to  distinguish  it 
from  pancreatic  amylase  (amyl opsin)  and  vegetable  amylases  (di- 
astase, etc.).  According  to  the  same  system,  the  fat-splitting 
enzyme  of  the  gastric  juice  would  be  termed  gastric  lipase  to  dif- 
ferentiate it  from  pancreatic  lipase  (steapsin),  the  fat  splitting 
enzyme  of  the  pancreatic  juice. 

Our  knowledge  regarding  the  distribution  of  enzymes  has  been 
wonderfully  broadened  in  recent  years.     Up  to  within  a  few  years, 

1  Produced  by  the  passage  of  electric  sparks  between  two  platinum  terminals 
immersed  in  distilled  water,  thus  liberating  ultra-microscopic  particles. 

2  Bredig  has  been  able  to  obtain  certain  inorganic  catalyzers  in  colloidal 
solution.    These  he  calls  "inorganic  enzymes." 

3  Substance  acted  upon. 

4  Armstrong  suggests  the  use  of  the  termination  "  clastic  "  instead  of  "  lytic." 
He  calls  attention  to  the  fact  that  amylolytic}  in  analogy  with  electrolytic,  means 
"  decomposition  by  means  of  starch "  and  is  therefore  a  misnomer.  He  sug- 
gests the  use  of  aniyloclastic,  protco clastic,  etc. 


4  PHYSIOLOGICAL    CHEMISTRY. 

the  real  scientific  information  as  to  the  enzymes  of  the  animal 
organism,  for  example,  was  limited,  in  the  main,  to  a  rather  crude 
understanding  of  the  enzymes  intimately  connected  with  the  main 
digestive  functions  of  the  organism.  We  now  have  occasion  to 
believe  that  enzymes  are  doubtless  present  in  every  animal  cell  and 
are  actively  associated  with  all  vital  phenomena.  As  a  preeminent 
example  of  such  cellular  activity  may  be  cited  the  liver  cell  with 
its  reputed  complement  of  15-20  or  more  enzymes. 

In  text-book  discussions  of  the  enzymes  it  is  customary  to  say 
that  very  little  is  known  regarding  the  chemical  characteristics  of 
these  substances  since  no  member  of  the  enzyme  group  has,  up  to 
the  present  time,  been  prepared  in  an  absolutely  pure  condition. 
Apparently,  however,  from  the  nature  of  the  facts  in  the  case,  it 
would  be  much  more  accurate  to  say  that  we  absolutely  do  not  knozv 
whether  a  specific  enzyme  lias,  or  has  not,  been  prepared  in  a  pure 
state.  (Some  authors,  like  Arthus,  have  assumed  that  enzymes 
are  not  chemical  individuals,  but  properties  conferred  upon  bodies.) 
The  enzymes  are  very  difficult  to  prepare  in  anything  like  a  con- 
dition approximating  purity,  since  they  are  very  prone  to  change 
their  nature  during  the  process  by  which  the  investigator  is  attempt- 
ing to  isolate  them.  For  this  reason  we  have  absolutely  no  proof 
that  the  final  product  obtained  is,  or  is  not,  in  the  same  state  of 
purity  it  possessed  in  the  original  cell.  Some  of  the  enzymes  are 
more  or  less  closely  associated  with  the  proteins  from  the  fact  that 
they  are  both  formed  in  every  cell  as  the  result  of  the  cellular  acti- 
vity, both  may  be  removed  from  solution  by  "  salting-out,"  both 
are  for  the  most  part  non-diffusible  and  are  probably  very  similar 
as  regards  elementary  composition.  Hence  in  the  preparation  of 
some  enzymes  it  is  extremely  difficult  to  make  an  absolute  separation 
from  the  protein.1  Under  certain  conditions  enzymes  are  readily 
adsorbed  by  shredded  protein  material,  such  as  fibrin,  and  may 
successfully  resist  the  most  prolonged  attempts  at  washing  them 
free.  We  may  summarize  some  of  the  properties  of  the  great 
body  of  enzymes  as  follows  :  Enzymes  are  soluble  in  dilute  glycerol, 
sodium  chloride  solution,  dilute  alcohol  and  water,  and  precipitable 
by  ammonium  sulphate  and  strong  alcohol.  Their  presence  may 
be  proven  from  the  nature  of  the  end-products  of  their  action  and 
not  through  the  agency  of  any  chemical  test.  They  are  colloidal 
and  non-diffusible ,and  occur  closely  associated  with  protein  material 
with  which  they  possess  many  properties  in  common.    Each  enzyme 

1  Others  seem  to  be  like  the  substrate  on  which  they  act,  e.  g.,  carbohydrate. 


ENZYMES    AND    THEIR    ACTION.  5 

shows  the  greatest  activity  at  a  certain  temperature  called  the 
optimum  temperature;  there  is  also  a  minimum  and  a  maximum 
temperature  for  each  specific  enzyme.  Their  action  is  inhibited  by 
sufficiently  lowering  the  temperature,  and  the  enzyme,  if  in  solution, 
is  entirely  destroyed  by  subjecting  it  to  a  temperature  of  ioo°  C. 
The  best  known  enzymes,  whether  derived  from  warm-blooded  or 
cold-blooded  animals,  are  most  active  between  35°-45°  C.  The 
nature  of  the  surrounding  media  alters  the  velocity  of  the  enzymatic 
action,  some  enzymes  being  more  active  in  acid  solution  whereas 
others  require  an  alkaline  fluid. 

Many  of  the  more  important  enzymes  do  not  occur  preformed 
within  the  cell,  but  are  present  in  the  form  of  a  zymogen  or  mother- 
substance.  In  order  to  yield  the  active  enzyme  this  zymogen  must 
be  transformed  in  a  certain  specific  manner  and  by  a  certain  specific 
substance.  This  transformation  of  the  inactive  zymogen  into  the 
active  enzyme  is  termed  activation.  For  instance,  the  zymogen  of 
the  enzyme  pepsin  of  the  gastric  juice,  termed  pepsinogen,  is  acti- 
vated by  the  hydrochloric  acid  secreted  by  the  gastric  cells  ( see 
p.  119),  whereas  the  activation  of  the  trypsinogen  of  the  pancreatic 
juice  is  brought  about  by  a  substance  termed  entcrokinasc1  (  see 
p.  141).  These  are  examples  of  many  well  known  activation  pro- 
cesses going  on  continually  within  the  animal  organism.  The 
ag'ency  which  is  instrumental  in  activating  a  zymogen  is  generally 
termed  a  zymo-excitcr  or  a  kinase.  In  the  cases  cited  hydrochloric 
acid  would  be  termed  a  zymo-exciter  and  enterokinase  would  be 
termed  a  kinase. 

After  filtering  yeast  juice,  prepared  by  the  Buchner  process  (see 
p.  2),  through  a  Martin  gelatin  filter.  Harden  and  Young  showed 
that  the  colloids  left  behind  and  the  filtrate  were  both  inactive 
fermentatively.  Upon  treating  the  colloid  material  (enzyme)  with 
some  of  the  filtrate,  however,  the  mixture  was  shown  to  be  able  to 
bring  about  pronounced  fermentation.  It  is  believed  that  a  co- 
enzyme present  in  the  filtrate  was  the  efficient  agent  in  the  trans- 
formation of  the  inactive  enzyme.  It  is  necessary  to  make  frequent 
renewals  of  the  co-enzyme  in  order  to  maintain  continuous  fermen- 
tation. It  was  further  shown  that  this  co-enzyme,  in  addition  to 
being  diffusible,  was  not  destroyed  by  boiling  and  that  it  disappeared 
from  yeast  juice  when  this  latter  was  fermented  or  allowed  to 
undergo  autolysis.     The  exact  nature  of  this  co-enzyme  of  zymase 

1  According  to  Delezenne  trypsinogen  ma}-  be  rapidly  activated  by  soluble 
calcium  salts. 


6  PHYSIOLOGICAL    CHEMISTRY. 

is  unknown.  The  co-enzyme  action,  in  this  case,  is  probably  de- 
pendent upon  the  presence  of  two  individual  agencies,  one  of  which' 
is  phosphates. 

It  has  been  shown  by  Loevenhart  that  the  property  of  acting 
as  a  pancreatic  lipase  co-enzyme  is  vested  in  bile  salts,  and  Magnus 
has  further  shown  that  the  synthetic  salts  are  as  efficient  in  this 
regard  as  the  natural  ones.  A  few  other  instances  of  co-enzyme 
demonstrations  have  been  reported. 

The  so-called  "  specificity "  of  enzyme  action  is  an  interesting 
and  important  fact.  That  enzymes  are  very  specific  as  to  the 
character  of  the  substrate  or  substance  acted  upon,  is  well  known. 
Emil  Fischer  investigated  this  problem  of  specificity  extensively 
in  connection  with  the  fermentation  of  sugars  and  reached  the 
conclusion  that  enzymes,  with  the  possible  exception  of  certain  oxi- 
dases, can  act  only  upon  such  substances  as  have  a  specific  stereo- 
isomeric  relationship  to  themselves.  He  considers  that  the  enzyme 
and  its  substrate  must  have  an  interrelation,  such  as  the  key  has 
to  the  lock,  or  the  reaction  does  not  occur.  Fischer  was  able  to 
predict,  in  certain  definite  cases,  from  a  knowledge  of  the  consti- 
tition  and  stereo-chemical  relationships  of  a  substance,  whether 
or  not  it  would  be  acted  upon  by  a  certain  enzyme.  An  applica- 
tion of  this  specificity  of  enzyme  action  may  be  seen  in  the  well- 
known  facts  that  certain  enzymes  act  on  carbohydrates,  others  on 
fats,  and  others  on  protein,  and  moreover,  that  the  group  of  those 
which  transform  carbohydrates,  for  example,  is  further  subdivided 
into  specific  enzymes  each  of  which  has  the  power  of  acting  alone 
upon  some  one  sugar.' 

It  has  been  conclusively  shown,  in  the  case  of  certain  enzymes,1 
at  least,  that  their  action  is  a  reversible  one  and  is,  in  all  its  main 
features,  directly  analogous  to  the  reversible  reactions  produced  by 
chemical  means.  For  instance,  in  the  saponification  of  ethyl-buty- 
rate  by  means  of  pancreatic  lipase,  it  has  been  shown  that  upon  the 
formation  of  the  end-products  of  the  reaction,  i.  e.,  butyric  acid 
and  ethyl  alcohol,  there  is  reversion2  and  the  reaction  is  stationary. 
This  does  not  mean  that  there  are  no  chemical  changes  going  on, 
but  simply  indicates  that  chemical  equilibrium  has  been  established, 

1  This  is  probably  a  general  condition. 

2  The  re-synthesis  of  ethyl-butyrate  from  its  hydrolysis  products.  This  may  be 
indicated  thus : 

CsHtCOOGH,  +  H.O  <=*  C3HrCOOH  +  C2H5OH. 

Ethyl  butyrate.  Butyric  acid.         Ethyl  alcohol. 


ENZYMES    AND    THEIR    ACTION.  / 

and  that  the  change  in  one  direction  is  counterbalanced  by  the 
change  in  the  opposite  direction.  Pancreatic  lipase  was  one  of  the 
first  enzymes  to  have  the  reversibility  of  its  reaction  clearly  demon- 
strated.1 A  knowledge  of  the  fact  that  lipase  possesses  this  rever- 
sibility of  action  is  of  extreme  physiological  importance  and  aids 
us  materially  in  the  explanation  of  the  processes  involved  in  the 
digestion,  absorption  and  deposition  of  fats  in  die  animal  organ- 
ism (see  p.  133). 

In  respect  to  many  enzymes  it  has  been  found  that  the  law  gov- 
erning the  action  of  inorganic  catalyzers  is  directly  applicable,  i.  e., 
that  the  intensity  is  almost  directly  proportional  to  the  concentra- 
tion of  the  enzyme.  In  the  case  of  enzymes,  however,  there  is  a 
difference  in  that  a  maximum  intensity  is  soon  reached  and  that 
subsequent  concentration  of  the  enzyme  is  productive  of  no  further 
increase  in  intensity.  The  enzymes  which  have  been  shown  to 
obey  this  linear  lazv  are  lipase,  invertase,  rennin  and  trypsin.  In 
certain  instances,  where  this  law  of  direct  proportionality  between 
the  intensity  of  action  and  the  concentration  of  enzyme  does  not 
hold,  it  has  been  found  that  the  law  of  Schiitz,  first  experimentally 
demonstrated  by  E.  Schiitz,  was  applicable.  This  is  to  the  effect 
that  the  intensity  is  directly  proportional  to  the  square  root  of 
the  concentration,  or  conversely,  that  the  relative  concentrations  of 
enzymes  are  directly  proportional  to  the  squares  of  the  intensities.2 

It  has  been  shown  that  there  are  certain  substances  which  possess 
the  property  of  directly  inhibiting  or  preventing  the  action  of  a 
catalyzer.  These  are  called  anti-catalyzers  or  paralyzers  and  have 
been  compared  to  the  anti-toxins.  Related  to  this  class  of  anti- 
catalytic  agents  stand  the  anti-enzymes.  The  first  anti-enzyme  to 
be  reported  was  the  anti-rennin  of  Morgenroth.  This  was  pro- 
duced by  injecting  into  an  animal  increasing  doses  of  rennet  solu- 
tion, whereupon  an  "  anti "  substance  was  subsequently  found  both 
in  the  serum  and  in  the  milk,  which  prevented  the  enzyme  rennin 
from  exerting  its  normal  activity  in  the  presence  of-  caseinogen. 
In  other  words,  anti-rennin  had  been  formed  in  the  serum  of  the 
animal,3  through  the  repeated  injections  of  rennet  solution.  Since 
the  discovery  of  this  anti-enzyme,  anti-bodies  have  been  demon- 
strated for  pepsin,  trypsin,  lipase,  urease,  amylase,  laccase,  tyro- 
sinase, emulsin,   papain,   and  thrombin.      According  to  Weinland, 

1  The  principle  was  first  demonstrated  in  connection  with  the  enzyme  maltase 
(see  p.  55). 

2  This  law   of  Schiitz  is  not  generally  applicable. 

3  Serum  is  normally  anti-try ptic. 


5  PHYSIOLOGICAL    CHEMISTRY. 

the  reason  why  the  stomach  does  not  digest  itself  is,  that  during 
life  there  is  present  in  the  mucous  membrane  of  the  stomach  an 
anti-enzyme  {anti-pepsin')  which  has  the  property  of  inhibiting  the 
action  of  pepsin.  A  similar  substance  (anti-trypsin)  is  present  in 
the  intestinal  mucosa  as  well  as  in  the  tissues  of  various  intestinal 
worms.  Some  investigators  are  not  inclined  to  accept  the  enzyme 
nature  of  these  inhibitory  agents  as  proven. 


EXPERIMENTS   ON   ENZYMES  AND  ANTI-ENZYMES 
A.    Experiments  on  Enzymes.1 

I.     AMYLASES. 

i.  Demonstration  of  Salivary  Amylase.2 — To  25  c.c.  of  a  one 
per  cent  starch  paste  in  a  small  beaker,  add  5  drops  of  saliva  and 
stir  thoroughly.  At  intervals  of  a  minute  remove  a  drop  of  the  so- 
lution to  one  of  the  depressions  of  a  test-tablet  and  test  by  the  iodine 
test.3  If  the  blue  color  with  iodine  still  forms  after  five  minutes, 
add  another  five  drops  of  saliva.  The  opalescence  of  the  starch 
solution  should  soon  disappear,  indicating  the  formation  of  soluble 
starch  (aniidulin)  which  gives  a  blue  color  with  iodine.  This 
body  should  soon  be  transformed  into  erythrodextrin  which  gives  a 
red  color  with  iodine  and  this,  in  turn,  should  pass  into*  achroodex- 
trin  which  gives  no  color  with  iodine.  This  point  is  called  the 
achromic  point.  When  this  point  is  reached  test  by  Fehling's  test4 
to  show  the  production  of  a  reducing  substance  (maltose).  A  posi- 
tive Fehling's  test  may  be  obtained  while  the  solution  still  reacts  red 
with  iodine  inasmuch  as  some  sugar  is  formed  from  the  soluble 
starch  coincidently  with  the  formation  of  the  erythrodextrin.  For 
further  discussion  of  the  transformation  of  starch  see  p.  54. 

2.  Demonstration  of  Pancreatic  Amylase.5 — Proceed  exactly 
as  indicated  above  in  the  Demonstration  of  Salivary  Amylase  ex- 
cept that  the  saliva  is  replaced  by  5  c.c.  of  pancreatic  extract  pre- 
pared as  described  on  p.  144.  Pancreatic  amylase  transforms  the 
starch  in  a  manner  entirelv  analogous  to  the  transformation  result- 
ing  from  the  action  of  salivary  amylase. 

1If  it  is  deemed  advisable  by  the  instructor  to  give  all  the  practical  work 
upon  enzymes  at  this  point  in  the  course,  additional  experiments  will  be  found 
in  Chapters  III,  VI  and  VIII. 

2  For  a  discussion  of  this    enzyme  see  p.   54. 

3  See  p.  44. 
*  See  p.  27. 

5  For  a   discussion  of  this  enzyme  see  p.   142. 


ENZYMES    AND    Till: Ik    ACTION.  9 

3.  Preparation  of  Vegetable  Amylase. —  Extracl  finely  ground 
malt  with  water,  filter  and  subject  the  filtrate  to  alcoholic  fermenta- 
tion by  means  of  yeast.  When  fermentation  is  complete  filter  off 
the  yeast  and  precipitate  the  amylase  from  the  filtrate  by  the  addi- 
tion of  alcohol.  The  precipitate  may  he  filtered  off  and  obtained  in 
the  form  of  a  fine  white  powder. 

4.  Demonstration  of  Vegetable  Amylase. — This  enzyme  may 
be  demonstrated  according  to  the  directions  given  under  Demonstra- 
tion of  Salivary  Amylase,  p.  8,  with  the  exception  that  the  saliva 
used  in  that  experiment  is  replaced  by  an  aqueous  solution  of  the 
vegetable  amylase  powder  prepared  as  described  above.1 

II.     PROTEASES. 

i.  Preparation  of  Gastric  Protease.2 — Treat  the  finely  com- 
minuted mucosa  of  a  pig's  stomach  with  0.4  per  cent  hydrochloric 
acid  and  extract  at  380  C.  for  24-48  hours.  The  filtrate  from 
this  mixture  constitutes  a  very  satisfactory  acid  extract  of  gastric 
protease  (see  p.  122). 

2.  Demonstration  of  Gastric  Protease. — Introduce  some  pro- 
tein material  (fibrin,  coagulated  egg-white,  or  washed  lean  beef) 
into  the  acid  extract  of  gastric  protease  prepared  as  above  described.3 
add  an  equal  volume  of  0.4  per  cent  hydrochloric  acid  and  place  the 
mixture  at  38 °  C.  for  2-3  days.  Identify  the  products  of  digestion 
according  to  directions  given  on  p.   122. 

3.  Preparation  of  Pancreatic  Protease.4 — A  satisfactory  ex- 
tract of  this  enzyme  may  be  made  from  the  pancreas  of  a  pig  or 
sheep  according  to  the  directions  given  on  p.  144. 

4.  Demonstration  of  Pancreatic  Protease. — Into  an  alkaline 
extract  of  pancreatic  protease,5  prepared  as  directed  on  p.  144.  in- 
troduce some  fibrin,  coagulated  egg-white  or  lean  beef  and  place 
the  mixture  at  380  C.  for  2-5  days.0     At  the  end  of  that  period 

1  If  desired  the  first  aqueous  extract  of  the  original  malt  may  be  used  in  this 
demonstration.     Commercial  taka-diastase  may  also  be  employed. 

2  Also  called  pepsin,  pepsase,  gastric  proteinase,  and  acid  protease.  For  a  dis- 
cussion of  this  enzyme  see  p.  120. 

3  If  so  desired  a  solution  of  commercial  pepsin  powder  in  0.2  per  cent  hydro- 
chloric acid  may  be  substituted. 

4  Also  called  trypsin,  trypsasc.  pancreatic  proteinase  and  alkali  proteinase. 
For  a  discussion  of  this  enzyme  see  p.  141. 

5  A  0.25  per  cent  sodium  carbonate  solution  of  commercial  trypsin  may  bo 
substituted. 

6  A  few  c.c.  of  toluene  or  an  alcoholic  solution  of  thymol  should  be  added  to 
prevent  putrefaction. 


IO  PHYSIOLOGICAL    CHEMISTRY. 

separate  and  identify  the  end-products  of  the  action  of  pancreatic 
protease  according  to  the  directions  given  on  p.  145. 

5.  Demonstration  of  a  Vegetable  Protease. — A  commercial 
preparation  of  papain  (papayotin,  carase  or  papase) ,  the  protease 
of  the  fruit  of  the  pawpaw  (carica  papaya),  may  be  used  in  this 
connection.  Follow  the  same  procedure  as  that  described  under 
gastric  protease  (see  p.  9). 

III.     LIPASES. 

i.  Preparation  of  Pancreatic  Lipase.1 — An  extract  of  this  en- 
zyme may  be  prepared  from  the  pancreas  of  the  pig  or  sheep  ac- 
cording to  the  directions  given  on  p.  144.2 

2.  Demonstration  of  Pancreatic  Lipase. — Into  each  of  two 
test-tubes  introduce  10  c.c.  of  milk  and  a  small  amount  of  litmus 
powder.  To  the  contents  of  one  tube  add  3  c.c.  of  a  neutral  ex- 
tract of  pancreatic  lipase  and  to  the  contents  of  the  other  tube  add 
3  c.c.  of  a  boiled  neutral  extract  of  pancreatic  lipase.  Keep  the 
tubes  at  38 °  C.  and  watch  for  color  changes.  The  blue  color  of 
the  litmus  powder  will  gradually  give  place  to  a  red.  This  change 
in  the  color  of  the  litmus  from  blue  to  red  has  been  brought  about 
by  the  fatty  acid  which  has  been  produced  through  the  lipolytic  ac- 
tion exercised  by  the  lipase  upon  the  milk  fats. 

3.  Preparation  of  Vegetable  Lipase. — This  enzyme  may  be 
readily  prepared  from  castor  beans,  several  months  old,  by  the  fol- 
lowing procedure  :3  Grind  the  shelled  beans'  very  fine4  and  ex- 
tract for  twenty-four  hour  periods  with  alcohol-ether  and  ether,  in 
turn.  Reduce  the  semi-fat-free  material  to  the  finest  possible  con- 
sistency by  means  of  mortar  and  pestle  and  pass  this  material 
through  a  sieve  of  very  fine  mesh.  Place  this  material  in  a  Soxhlet 
extractor  and  extract  for  one  week.  This  fat-free  powder  may 
then  be  used  to  demonstrate  the  action  of  vegetable  lipase.  Powder 
prepared  as  described  may  be  used  in  quantitative  tests.  For  ordi- 
nary qualitative  tests  it  is  not  necessary  to  remove  the  last  traces 
of  fat  and  therefore  the  extraction  period  in  the  Soxhlet  apparatus 
may  be  much  shortened. 

1  Also  called  steapsin.  For  a  discussion  of  this  enzyme  see  p.  143.  A  very 
active  lipolytic  extract  may  also  be  prepared  from  the  liver. 

2  If  preferred  a  glycerol  extract  may  be  prepared  according  to  the  directions 
given  by  Kanitz ;  Zeitschrift  fur  physiologische  Chemie,  1906,  XLVI,  p.  482. 

3  A.  E.  Taylor:  On  Fermentation;  University  of  California  Publications,  1907. 
1  The  shells  should  be  removed  without  the  use  of  water.     These  beans  are 

poisonous  due  to  their  content  of  ricin. 


ENZYMES    AND    THEIR    ACTION.  I  I 

4.  Demonstration  of  Vegetable  Lipase. — The  lipolytic  action 
of  the  lipase  prepared  from  the  castor  bean,  as  just  described,  may 
be  demonstrated  in  a  manner  entirely  analogous  to  that  used  in  the 
Demonstration  of  Pancreatic  Lipase,  see  p.  10.  Proceed  as  indi- 
cated in  that  experiment  and  substitute  the  vegetable  lipase  powder 
for  the  neutral  extract  of  pancreatic  lipase.  The  type  of  action  is 
entirely  analogous  in  the  two  instances. 

An  experiment  similar  to  Experiment  2,  p.  149,  may  also  be  tried 
if  desired.  In  this  experiment  0.2  c.c.  of  either  ethyl  batyrate  or 
aniyl  acetate  may  be  employed. 

IV.     INVERTASES.1 

i.  Preparation  of  an  Extract  of  Sucrase.2 — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  dog,  pig,  rat,  rabbit,  or 
hen,  with  about  three  volumes  of  a  two  per  cent  solution  of  sodium 
fluoride  and  permit  the  mixture  to  stand  at  room  temperature  for 
twenty-four  hours.  Strain  the  extract  through  cloth  or  absorbent 
cotton  and  use  the  strained  material  in  the  following  demonstra- 
tion. 

2.  Demonstration  of  Sucrase. — To  about  5  c.c.  of  a  one  per 
cent  solution  of  sucrose,  in  a  test-tube,  add  about  one  cubic  centi- 
meter of  a  two  per  cent  sodium  fluoride  intestinal  extract,  prepared 
as  described  above.  Prepare  a  control  tube  in  which  the  intestinal 
extract  is  boiled  before  being  added  to  the  sugar  solution.  Place 
the  two  tubes  at  380  C.  for  two  hours.3  Heat  the  mixture  to  boil- 
ing to  coagulate  the  protein  material,  filter,  and  test  the  filtrate  by 
Fehling's  test  (see  p.  27).  The  tube  containing  the  boiled  extract 
should  give  no  response  to  Fehling's  test  whereas  the  tube  con- 
taining the  unboiled  extract  should  reduce  the  Fehling's  solution. 
This  reduction  is  due  to  the  formation  of  invert  sugar  (see  p.  41  ^ 
from  the  sucrose  through  the  action  of  the  enzyme  sucrase  which 
is  present  in  the  intestinal  epithelium. 

3.  Preparation  of  Vegetable  Sucrase. — Thoroughly  grind 
about  100  grams  of  brewer's  yeast  in  a  mortar  with  sand.  Spread 
the  ground  yeast  in  thin  layers  on  glass  or  porous  plates  and  dry 
it  rapidly  in  a  current  of  dry,  warm  air.  Powder  this  dry  yeast, 
extract  it  with  distilled  water  and  filter.     Pour  the  filtrate  into 

1  The  inverting  enzymes  of  the  alimentary  tract;  Mendel  and  Mitchell : 
American  Journal  of  Physiology,  1907-08,  XX,  p.  81. 

2  For  a  discussion  of  this  enzyme  see  p.  144. 

8  If  a  positive  result  is  not  obtained  in  this  time  permit  the  digestion  to 
proceed  for  a  longer  period. 


12  PHYSIOLOGICAL    CHEMISTRY. 

acetone,  stir  and  after  permitting  the  acetone  mixture  to  stand  for 
a  few  minutes  filter  on  a  Buchner  funnel.  The  resulting  precipi- 
tate, after  drying  and  pulverizing,  may  be  used  to  demonstrate 
vegetable  sucrase. 

4.  Demonstration  of  Vegetable  Sucrase. — To  about  5  c.c.  of  a 
one  per  cent  solution  of  sucrose  in  a  test-tube  add  a  small  amount 
of  the  sucrase  powder  prepared  as  directed  above.  Place  the  tube 
at  380  C.  for  24-72  hours  and  at  the  end  of  that  period  test  the 
solution  by  Fehling's  test.  Reduction  indicates  that  the  active 
sucrase  powder  has  transformed  the  non-reducing  sucrose  into 
dextrose  and  lsevulose,  and  these  sugars,  in  turn,  have  reduced  the 
Fehling  solution. 

5.  Preparation  of  an  Extract  of  Lactase.1 — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  kitten,  puppy,  or  pig 
embryo  with  about  three  volumes  of  a  two  per  cent  solution  of 
sodium  fluoride  and  permit  the  mixture  to  stand  at  room  tempera- 
ture for  twenty- four  hours.  Strain  the  extract  through  cloth  or 
absorbent  cotton  and  use  the  strained  material  in  the  following 
demonstration. 

6.  Demonstration  of  Lactase.2 — To  about  5  c.c.  of  a  one  per 
cent  solution  of  lactose  in  a  test-tube  add  about  one  cubic  centi- 
meter of  a  toluene-water  extract  or  a  two  per  cent  sodium  fluoride 
extract  of  the  first  part  of  the  small  intestine3  of  a  kitten,  puppy,  or 
pig  embryo  prepared  as  described  above.  Prepare  a  control  tube 
in  which  the  intestinal  extract  is  boiled  before  being  added  to  the 
sugar  solution.  Place  the  two  tubes  at  38  °  C.  for  24  hours.  At 
the  end  of  this  period  add  one  cubic  centimeter  of  the  digestion 
mixture  to  5  c.c.  of  Barfoed's4  reagent  and  place  the  tubes  in  a 
boiling  water-bath.5  Examine  the  tubes  at  the  end  of  three  min- 
utes against  a  black  background  in  a  good  light.  If  no  cuprous 
oxide  is  visible  replace  the  tubes  and  repeat  the  examination  at  the 
end  of  the  fourth  and  fifth  minutes.  If  no  reduction  is  then  ob- 
served permit  the  tubes  to  stand  at  room  temperature  for  5-10  min- 
utes and  examine  again.6 

1  For  a  discussion   of  this  enzyme  see  p.   144. 
3  Roaf ;  Bio-Chemical  Journal,  1908,  III,  p.   182. 

3  Duodenum  and  first  part  of  jejunum. 

4  To  4.5  grams  of  neutral  crystallized  cupric  acetate  in  900  c.c.  of  water,  add 
0.6  c.c.  of  glacial  acetic  acid  and  make  the  total  volume  of  the  solution  one  liter. 

5  Care  should  be  taken  to  see  that  the  water  in  the  bath  reaches  at  least  to  the 
upper  level  of  the  contents  of  the  tubes. 

6  Sometimes  the  drawing  of  conclusions  is  facilitated  by  pouring  the  mixture 
from  the  tube  and  examining  the  bottom  of  the  tube  for  adherent  cuprous  oxide. 


ENZYMES    AND    THEIR    ACTION.  13 

It  has  been  determined  that  disaccharide  solutions  will  not 
reduce' Barf oed's  reagent  until  after  they  have  been  heated  for  9-10 
minutes  on  a  boiling-  water-bath  in  contact  with  the  reagent.1 
Therefore  in  the  above  test,  if  the  tube  containing  the  unboiled 
extract  exhibits  any  reduction  after  being  heated  as  indicated,  for 
a  period  of  five  minutes  or  less,  and  the  control  tube  containing 
boiled  extract  shows  no  reduction,  it  may  be  concluded  that  lactase 
was  present  in  the  intestinal  extract.2 

7.  Preparation  of  an  Extract  of  Maltase.3 — Treat  the  finely 
divided  epithelium  of  the  small  intestine  of  a  cat,  kitten,  or  pig 
(embryo  or  adult)  with  about  three  volumes  of  a  two  per  cent  solu- 
tion of  sodium  fluoride  and  permit  the  mixture  to  stand  at  room 
temperature  for  twenty-four  hours.  Strain  the  extract  through 
cloth  and  use  the  strained  material  in  the  following  demonstration. 

8.  Demonstration  of  Maltase. — Proceed  exactly  as  indicated  in 
the  demonstration  of  lactase,  above,  except  that  a  one  per  cent  solu- 
tion of  maltose  is  substituted  for  the  lactose  solution.  The  extract 
used  may  be  prepared  from  the  upper  part  of  the  intestine  of  a  cat, 
kitten,  or  pig  (embryo  or  adult).  In  the  case  of  lactase,  as  indi- 
cated, the  intestine  used  should  be  that  of  a  kitten,  puppy,  or  pig 
(embryo). 

V.  EREPSIN.4 

1.  Preparation  of  Erepsin. — Grind  the  mucous  membrane  of 
the  small  intestine  of  a  cat,  dog  or  pig,  with  sand  in  a  mortar. 
Treat  the  mortared  membrane  with  toluene-  or  chloroform-water 
and  permit  the  mixture  to  stand,  with  occasional  shaking,  for  24-72 
hours.5  Filter  the  extract  thus  prepared  through  cotton  and  use 
the  filtrate  in  the  following  experiment. 

2.  Demonstration  of  Erepsin. — To  about  5  c.c.  of  a  one 
per  cent  solution  of  Witte's  peptone  in  a  test-tube -add  about  one 
c.c.  of  the  erepsin  extract  prepared  as  described  above  and  make 
the  mixture  slightly  alkaline  (0.1  per  cent)  with  sodium  carbonate. 
Prepare  a  second  tube  containing  a  like  amount  of  peptone  solu- 
tion but  boil  the  erepsin  extract  before  introducing  it.     Place  the 

xThe  heating  for  9-10  minutes  is  sufficient  to  transform  the  disaccharide  into 
monosaccharide. 

2  The  reduction  would,  of  course,  be  due  to  the  action  of  the  dextrose  and 
galactose  which  had  been  formed  from  the  lactose  through  the  action  of  the 
enzyme  lactase. 

3  For  a   discussion  of    this  enzyme  see  p.   55. 

4  Also  called  erepsase.     For  a  discussion  of  this  enzyme  see  p.   143. 

5  The  enzyme  may  also  be  extracted  by  means  of  glycerol  or  alkaline  "physi- 
ological" salt  solution  if  desired. 


14  PHYSIOLOGICAL    CHEMISTRY. 

two  tubes  at  380  C.  for  2-3  days.  At  the  end  of  that  period  heat 
the  contents  of  each  tube  to  boiling,  filter  and  try  the  biuret  test 
on  each  filtrate.  In  making  these  tests  care  should  be  taken  to  use 
like  amounts  of  filtrate,  potassium  hydroxide  and  cupric  sulphate 
in  each  test  in  order  that  the  drawing  of  correct  conclusions  may  be 
facilitated.  The  contents  of  the  tube  which  contained  the  boiled 
extract  should  show  a  deep  pink  color  with  the  biuret  test,  due  to 
the  peptone  still  present.  On  the  other  hand  the  biuret  test  upon 
the  contents  of  the  tube  containing  the  unboiled  extract  should  be 
negative  or  exhibit,  at  the  most,  a  faint  pink  or  blue  color,  signify- 
ing that  the  peptone,  through  the  influence  of  the  erepsin,  has  been 
transformed,  in  great  part  at  least,  into  amino  acids  which  do  not  re- 
spond to  the  biuret  test.1 

VI.     URICOLYTIC   ENZYME.2 

1.  Preparation  of  Uricolytic  Enzyme. — Extract  pulped  liver 
tissue  with  toluene-  or  chloroform-water  at  380  C.  for  24  hours, 
with  occasional  shaking.  Filter  the  extract  and  use  the  filtrate  in 
the  following  experiment. 

2.  Demonstration  of  Uricolytic  Enzyme. — Add  about  0.1 
gram  of  uric  acid  to  10  c.c.  of  water  and  bring  the  uric  acid  into 
solution  by  the  addition  of  the  minimal  quantity  of  potassium  hy- 
droxide. To  5  c.c.  of  this  uric  acid  solution,  in  a  test-tube,  add  5 
c.c.  of  the  uricolytic  enzyme  extract  prepared  as  described  above. 
Prepare  a  second  tube  containing  a  like  amount  of  uric  acid  solution 
but  boil  the  extract  before  it  is  introduced.  Place  the  two  tubes 
at  38 °  C.  for  3-4  days  and  titrate  the  two  digestive  mixtures  with 
a  solution  of  potassium  permanganate  according  to  directions  given 
under  Folin-Shaffer  Method,  Chapter  XXII.  It  will  be  found  that 
the  mixture  containing  the  boiled  extract  requires  a  much  larger 
volume  of  the  permanganate  to  complete  the  titration  than  the  other 
tube.  This  indicates  that  a  uricolytic  enzyme  has  destroyed  at  least 
a  portion  of  the  uric  acid  which  was  originally  present  in  the  tube 
containing  the  unboiled  extract. 

VII.  CATALASE. 

Demonstration  of  Catalase. — The  various  animal  tissues,  such 
as  liver,  kidney,  blood,  lung,  muscle  and  brain  contain  an  enzyme 

1  Strictly  speaking  this  erepsin  demonstration  is  not  adequate  unless  a  control 
test  is  made  with  native  protein  (except  caseinogen,  histones  and  protamines) 
to  show  that  the  extract  is  trypsin-free  and  digests  peptone  but  not  native 
protein. 

2  Mendel  and  Mitchell ;  American  Journal  of  Physiology,  1908,  XX,  p.  97. 


ENZYMES    AND    THEIR    ACTION.  I  5 

called  caialase  which  possesses  the  property  of  decomposing  hydro- 
gen peroxide.  The  presence  of  this  enzyme  may  he  demonstrated 
as  follows:  Introduce  into  a  low,  broad,  wide-mouthed  bottle  some 
pulped  liver  tissue  and  a  porcelain  crucible  containing  neutral  hydro- 
gen peroxide.1  Connect  the  bottle  with  a  eudiometer  filled  with 
water,  upset  the  crucible  of  hydrogen  peroxide  upon  the  liver  pulp 
and  note  the  collection  of  gas  in  the  eudiometer.  This  gas  is  oxy- 
gen which  has  been  liberated  from  the  hydrogen  peroxide  through 
the  action  of  the  catalase  of  the  liver  tissue. 

B.     Experiments  on  Anti-Enzymes. 

1.  Preparation  of  an  Extract  of  Anti-Pepsin.2-— Grind  up  a 

number  of  intestinal  worms  (ascaris)3  with  quartz  sand  in  a  mortar. 
Subject  this  mass  to  high  pressure,  filter  the  resultant  juice  and 
treat  it  with  alcohol  until  a  concentration  of  sixty  per  cent  is  reached. 
If  any  precipitate  forms  it  should  be  filtered  off4  and  alcohol  added 
to  the  filtrate  until  the  concentration  of  alcohol  is  85  per  cent, 
or  over.  The  anti-enzyme  is  precipitated  by  this  concentration. 
Permit  this  precipitate  to  stand  for  twenty-four  hours,  then  filter 
it  off,  wash  it  with  95  per  cent  alcohol,  absolute  alcohol,  and  ether, 
in  turn,  and  finally  dry  the  substance  over  sulphuric  acid.  The 
sticky  powder  which  results  may  be  used  in  this  form  or  may  be 
dissolved  in  water  as  desired  and  the  aqueous  solution  used.5 

2.  Demonstration  of  Anti-Pepsin.6 — Introduce  into  a  test-tube 
a  few  fibrin  shreds  and  equal  volumes  of  pepsin-hydrochloric  acid7 
and  ascaris  extract  made  as  indicated  above.  Prepare  a  control  tube 
in  which  the  ascaris  extract  is  replaced  by  water.  Place  the  two 
tubes  at  380  C.  Ordinarily  in  one  hour  the  fibrin  in  the  control 
tube  will  be  completely  digested.  The  fibrin  in  the  tube  containing 
the  ascaris  extract  may,  however,  remain  unchanged  for  days,  thus 
indicating  the  inhibitory  influence  exerted  by  the  anti-enzyme  pres- 
ent in  this  extract. 

3.  Preparation  of  an  Extract  of  Anti-Trypsin.s — The  extract 

1  Mendel  and  Leavenworth;  American'  Journal  of  Physiology,  1908,  XXI,  p.  85. 
~  Anti-gastric-protease    or  anti-acid-protease. 

3  These  may  be  readily  obtained  from  pigs  at  a  slaughter  house. 

4  This  precipitate  consists  of  impurities,  the  anti-enzyme  not  being  precipitated 
until  a  higher  concentration  of  alcohol  is  reached. 

5  The  original  ascaris  extract  possesses  much  greater  activity  than  either  the 
powder  or  the  aqueous  solution. 

6  Martin  H.  Fischer;   Physiology  of  Alimentation,  1907,  p.   134. 

7  Made  by  bringing  0.015  gram  of  pepsin  into  solution  in  7  c.c.  of  water  and 
0.23  gram  of  concentrated  hydrochloric  acid. 

s  Anti-pancreatic-protease  or  Anti-alkali-protease. 


1 6  PHYSIOLOGICAL    CHEMISTRY. 

may  be  prepared  from  the  intestinal  worm,  ascaris,  according  to  the 
directions  given  on  page  15. 

4.  Demonstration  of  Anti-Trypsin. — Introduce  into  a  test-tube 
a  few  shreds  of  fibrin  and  equal  volumes  of  an  artificial  tryptic 
solution1  and  the  ascaris  extract  made  as  described  on  page  15. 
Prepare  a  control  tube  in  which  the  ascaris  extract  is  replaced  by 
water.  Place  the  two  tubes  at  38 °  C.  Ordinarily  the  fibrin  in  the 
control  tube  will  be  completely  digested  in  two  hours.  The  fibrin 
in  the  tube  containing  the  ascaris  extract  may,  however,  remain 
unchanged  for  days,  thus  indicating  the  inhibitory  influence  of  the 
anti-enzyme. 

Blood  serum  also  contains  anti-try p sin.  This  may  be  demon- 
strated as  follows :  Introduce  equal  volumes  of  serum  and  artificial 
tryptic  solution  (prepared  as  described  above)  into  a  test-tube  and 
add  a  few  shreds  of  fibrin.  Prepare  a  control  tube  containing  boiled 
serum.  Place  the  two  tubes  at  380  C.  It  will  be  observed  that  the 
fibrin  in  the  tube  containing  the  boiled  serum  digests,  whereas  that 
in  the  other  tube  does  not  digest.  The  anti-trypsin  present  in  the 
unboiled  serum  has  exerted  an  inhibitory  influence  upon  the  action 
of  the  trypsin. 

C.     Quantitative  Applications. 

1.  Quantitative  Determination  of  Amy loly  tic  Activity. — Wohl- 
gemuth^ Method. — Arrange  a  series  of  test-tubes  with  diminishing 
quantities  of  the  enzyme  solution  under  examination,  introduce  into 
each  tube  5  c.c.  of  a  1  per  cent  solution  of  soluble  starch2  and  place 
each  tube  at  once  in  a  bath  of  ice  water.3  When  all  the  tubes 
have  been  prepared  in  this  way  and  placed  in  the  ice  water-bath  they 
are  transferred  to  a  water-bath  or  incubator  and  kept  at  38 °  C. 
for  from  thirty  minutes  to  an  hour.4     At  the  end  of  this  digestion 

1  Made  by  dissolving  0.04  gram  of  sodium  carbonate  and  0.015  gram  of  trypsin 
in  8  c.c.  of  water. 

2  Kahlbaum's  soluble  starch  is  satisfactory.  In  preparing  the  1  per  cent  solu- 
tion, the  weighed  starch  powder  should  be  dissolved  in  the  proper  volume  of 
cold,  distilled  water  and  stirred  until  a  homogeneous  suspension  is  obtained. 
The  mixture  should  then  be  heated,  with  constant  stirring,  in  a  porcelain  dish, 
until  it  is  clear.  This  ordinarily  takes  about  8-10  minutes.  A  slightly  opaque 
solution  is  thus  obtained  which  should  be  cooled  before  using. 

3  Ordinarily  a  series  of  six  tubes  is  satisfactory,  the  volumes  of  the  enzyme 
solution  used  ranging  from  1  c.c.  to  0.1  c.c.  and  the  measurements  being  made 
by  means  of  a  1  c.c.  graduated  pipette.  Each  tube  should  be  placed  in  the  ice 
water  bath  as  soon  as  the  starch  solution  is  introduced.  It  will  be  found 
convenient  to  use  a  small  wire  basket  to  hold  the  tubes. 

4 Longer  digestion  periods  may  be  used  where  it  is  deemed  advisable.  If  ex- 
ceedingly weak  solutions  are  being  investigated,  it  may  be  most  satisfactory  to 
permit  the  digestion  to  extend  over  a  period  of  24  hours. 


ENZYMES    AND    THEIR    ACTION.  17 

period  the  tubes  are  again  removed  to  the  bath  of  ice  water  in 
order  that  the  action  of  the  enzyme  may  be  stopped. 

Dilute  the  contents  of  each  tube,  to  within  about  one-half  inch 
of  the  top,  with  water,  add  one  drop  of  a  T^  solution  of  iodine  and 
shake  the  tube  and  contents  thoroughly.  A  series  of  colors  rang- 
ing from  dark  blue  through  bluish-violet  and  reddish-yellow  to 
yellow,  will  be  formed.1  The  dark  blue  color  shows  the  presence 
of  unchanged  starch,  the  bluish-violet  indicates  a  mixture  of  starch 
and  erythrodextrin,  whereas  the  reddish-yellow  signifies  that  eryth- 
rodextrin  and  maltose  are  present,  and  the  yellow  solution  denotes 
the  complete  transformation  of  starch  into  maltose.  Examine  the 
tubes  carefully  before  a  white  background  and  select  the  last  tube 
in  the  series  which  shows  the  entire  absence  of  all  blue  color,  thus 
indicating  that  the  starch  has  been  completely  transformed  into 
dextrins  and  sugar.  In  case  of  indecision  between  two  tubes, 
add  an  extra  drop  of  the  iodine  solution,  and  observe  them  again, 
after  shaking, 

Calculation. — The  amylolytic  activity2  of  a  given  solution  is 
expressed  in  terms  of  the  activity  of  i  c.c.  of  such  a  solution.  For 
example,  if  it  is  found  that  0.02  c.c.  of  an  amylolytic  solution, 
acting  at  380  C.  completely  transformed  the  starch  in  5  c.c.  of  a 
1  per  cent  starch  solution  in  30  minutes,  the  amylolytic  activity  of 
such  a  solution  would  be  expressed  as  follows : 

<  =  25o. 

This  indicates  that  1  c.c.  of  the  solution  under  examination  pos- 
sesses the  power  of  completely  digesting  250  c.c.  of  a  1  per  cent 
starch  solution  in  30  minutes  at  38 °  C. 

2.  Quantitative  Determination  of  Peptic  Activity,  (a) 
Mett's  Method. — The  determination  of  the  actual  rate  of  peptic  ac- 
tivity is  a  most  important  procedure  under  certain  conditions.  Sev- 
eral methods  of  making  this  determination  are  in  use.  The  method 
of  Sprigg3  is  probably  the  most  accurate  method  yet  devised  for 
this  purpose.  It  is,  however,  too  complicated  and  time-consuming 
for  clinical  purposes.  The  method  of  Mett,  given  below,  is  very 
simple  although  not  strictly  accurate.  The  procedure  is  as  follows : 
To  about  5  c.c.  of  the  gastric  juice  under  examination  in  a  test- 

1  See  p.  44. 

'  Designated  by  "  D  "  the  first  letter  of  "  diastatic." 

3  Sprigg:  Zeitschrift  fur  physiologische  Chemie,  1902.  XXXV,  p.  465. 


1 8  PHYSIOLOGICAL    CHEMISTRY. 

tube  add  a  section  of  a  Mett  tube1  and  place  the  mixture  at  380  C. 
for  ten  hours.  At  the  end  of  this  period,  the  tube  should  be  re- 
moved from  the  gastric  juice  and  the  length  of  the  column  of 
coagulated  albumin  which  has  been  digested,  carefully  determined 
by  means  of  a  low  power  microscope  and  a  millimeter  scale.  In 
normal  human  gastric  juice  the  upper  limit  is  4  mm.  However, 
control  tests  should  always  be  made  to  determine  the  digestibility 
of  the  coagulated  albumin  in  artificial  gastric  juice  inasmuch  as 
this  factor  will  vary  with  different  albumin  preparations. 

In  connection  with  this  test  Schiitz's  law  should  be  borne  in 
mind.  This  principle  is  to  the  effect  that  the  amount  of  proteolytic 
enzyme  present  in  a  digestion  mixture  is  proportional  to  the  square 
of  the  number  of  millimeters  of  albumin  digested.  Therefore  a 
gastric  juice  which  digests  2  mm.  of  albumin  contains  four  times 
as  much  pepsin  as  a  gastric  juice  which  digests  only  1  mm.  of  albu- 
min. And  further,  if  the  quantities  of  albumin  digested  are  2  mm. 
and  3  mm.  respectively,  the  ratio  between  the  pepsin  values  will 
be  as  4 : 9. 

It  is  claimed  by  Nirenstein  and  Schiff  that  the  principle  of 
Schutz  does  not  apply  to  gastric  juice  unless  this  fluid  be  diluted 
with  fifteen  volumes  of  N/20  hydrochloric  acid. 

(b)  Fuld  and  Levison's  Method. — This  test  is  founded  upon 
the  fact,  shown  by  Osborne,  that  edestin  when  brought  into  solu- 
tion in  dilute  acid  will  change  in  its  solubility,  due  to  the  contact 
with  the  acid,  and  that  a  protean  called  edestan,  which  is  insoluble 
in  neutral  fluid,  will  be  formed.  The  procedure  is  as  follows : 
Dilute  the  gastric  juice  under  examination  with  20  volumes  of 
water  and  introduce  gradually  decreasing  volumes  of  the  diluted 
juice  into  a  series2  of  narrow  test-tubes  about  1  cm.  in  diameter. 

1  In  the  preparation  of  these  tubes,  egg-white  is  diluted  with  an  equal  volume 
of  water,  the  precipitated  globulin  filtered  off  and  the  filtrate  collected  in  a 
tall,  narrow  beaker  or  a  large  test-tube.  A  bundle  of  capillary  tubes  about  10 
cm.  in  length  and  2  mm.  in  diameter  are  now  placed  in  this  vessel  in  such  a 
manner  that  they  are  completely  submerged  in  the  albumin  solution.  After  an 
examination  has  shown  that  the  tubes  are  completely  filled  with  the  albumin 
solution  and  that  there  are  no  interfering  air-bubbles,  the  vessel  and  its  con- 
tained tubes  is  heated  for  5-15  minutes  in  a  boiling  water-bath,  in  order  to 
coagulate  the  albumin.  When  this  coagulation  is  complete,  the  tubes  are  re- 
moved, all  albumin  adhering  to  them  is  carefully  cleaned  off,  and  the  tubes 
rendered  air-tight  by  the  application  of  sealing  wax  at  either  end.  When 
needed  for  use,  these  tubes  are  cut  into  sections  about  2  cm.  in  length. 

2  The  longer  the  series,  the  more  accurate  the  deductions  which  may  be  drawn. 


ENZYMES    AND    THEIR    ACTION.  19 

The  measurements  of  gastric  juice  may  conveniently  be  made  with 
a  one  c.c.  pipette  which  is  accurately  graduated  in  Moo  c.c.  Into 
the  first  tube  in  the  series  may  be  introduced  one  c.c.  of  gastric 
juice,  and  the  tubes  which  follow  in  the  series  may  receive  vol- 
umes which  differ,  in  each  instance,  from  the  volume  introduced 
into  the  preceding  tube  by  Moo,  Yso,  !•!<>•  or  Mo  of  a  cubic  centi- 
meter. Now  rapidly  introduce  into  each  tube  the  same  volume 
(e.  g.,  2  c.c.)  of  a  i  :  iooo  solution  of  edestin'1  and  place  the  tubes 
at  40 °  C.  for  one-half  hour.  At  the  end  of  this  time  stratify 
ammonium  hydroxide  upon  the  contents  of  each  tube,2  place  the 
tubes  in  position  before  a  black  background  and  examine  them 
carefully.  The  ammonium  hydroxide,  by  diffusing  into  the  acid 
fluid,  forms  a  neutral  zone  and  in  this  zone  will  be  precipitated  any 
undigested  edestan  which  is  present.  Select  the  tube  in  the  series 
which  contains  the  least  amount  of  gastric  juice  and  which  ex- 
hibits  no  ring,  signifying  that  the  edestan  has  been  completely 
digested,  and  calculate  the  peptic  activity  of  the  gastric  juice  under 
examination  on  the  basis  of  the  volume  of  gastric  juice  used  in  this 
particular  tube. 

Calculation. — Multiply  the  number  of  c.c.  of  edestin  solution 
used  by  the  dilution  to  which  the  gastric  juice  was  originally  sub- 
jected and  divide  the  volume  of  gastric  juice  necessary  to  com- 
pletely digest  the  edestan  by  this  product.  For  example,  if  2  c.c. 
of  the  edestin  solution  was  completely  digested  by  0.25  c.c.  of  a 
1  :  20  gastric  juice  we  would  have  the  following  expression ; 
0.25  -i-  20  X  2  or  1  :  160.  This  peptic  activity  may  be  expressed  in 
several  ways,  e.  g.,  (a)  1:160  pepsin;  (b)  160  pepsin  content; 
(c)   160  parts. 

3.  Quantitative  Determination  of  Tryptic  Activity. — Gross' 
Method. — This  method  is  based  upon  the  principle  that  faintly  alka- 
line solutions  of  casein  are  precipitated  upon  the  addition  of  dilute 
(1  per  cent)  acetic  acid  whereas  its  digestion  products  are  not  so 
precipitated.     The  method  follows :    Prepare  a  series  of  tubes  each 

1  This  edestin  should  be  prepared  in  the  usual  way  (see  p.  103),  and  brought 
into  solution  in  a  dilute  hydrochloric  acid  of  approximately  the  same  strength 
as  that  which  occurs  normally  in  the  human  stomach.  This  may  be  conveniently 
made  by  adding  30  c.c.  of  fT)  hydrochloric  acid  to  70  c.c.  of  water.  Ordinarily 
it  should  not  take  longer  than  one  minute  to  introduce  the  edestin  solution  into 
the  entire  series  of  tubes.  However,  if  the  edestin  is  added  to  the  tubes  in  the 
same  order  as  the  ammonium  hydroxide  is  afterward  stratified,  no  appreciable 
error  is  introduced. 

2  Making  the  stratification  in  the  same  order  as  the  edestin  solution  was  added. 


20  PHYSIOLOGICAL    CHEMISTRY. 

containing  10  c.c.  of  a  o.  i  per  cent  solution  of  pure,  fat-free,  casein,1 
which  has  been  heated  to  a  temperature  of  400  C.  Add  to  the 
contents  of  the  series  of  tubes  increasing  amounts  of  the  trypsin 
solution  under  examination,2  and  place  them  at  400  C.  for  fifteen 
minutes.  At  the  end  of  this  time  remove  the  tubes  and  acidify 
the  contents  of  each  with  a  few  drops  of  dilute  (1  per  cent)  acetic 
acid.  The  tubes  in  which  the  casein  is  completely  digested  will 
remain  clear  when  acidified  while  those  tubes  which  contain  undi- 
gested casein  will  become  more  or  less  turbid  under  these  condi- 
tions. Select  the  first  tube  in  the  series  which  exhibits  no  turbidity 
upon  acidification,  thus  indicating  complete  digestion  of  the  casein, 
and  calculate  the  tryptic  activity  of  the  enzyme  solution  under  ex- 
amination. 

Calculation. — The  unit  of  tryptic  activity  is  an  expression  of  the 
power  of  1  c.c.  of  the  fluid  under  examination  exerted  for  a  period 
of  fifteen  minutes  on  10  c.c.  of  a  0.1  per  cent  casein  solution.  For 
example,  if  0.5  c.c.  of  a  trypsin  solution  completely  digests  10  c.c. 
of  a  0.1  per  cent  solution  of  casein  iii  fifteen  minutes  the  activity  of 
that  solution  would  be  expressed  as  follows : 

Tryptic  activity  =  1  -f-  0.5  =  2. 

Such  a  trypsin  solution  would  be  said  to  possess  an  activity  of 
2.  If  0.3  c.c.  of  the  trypsin  solution  had  been  required  the  solution 
would  be  said  to  possess  an  activity  of  3.3  i.  e.,  1  -^-0.3  =  3.3. 

1  Made  by  dissolving  one  gram  of  Griibler's  casein  in  a  liter  of  0.1  per  cent 
sodium  carbonate.    A  little  chloroform  may  be  added  to  prevent  bacterial  action. 

2  The  amount  of  solution  used  may  vary  from  0.1-1  c.c.     The  measurements 
may  conveniently  be  made  by  means  of  a  1  c.c.  graduated  pipette. 


CHAPTER    II. 

CARBOHYDRATES. 

The  name  carbohydrates  is  given  to  a  class  of  bodies  which  are 
an  especially  prominent  constituent  of  plants  and  which  are  found 
also  in  the  animal  body  either  free  or  as  an  integral  part  of  various 
proteins.  They  are  called  carbohydrates  because  they  contain  the 
elements  C,  H  and  O;  the  H  and  O  being  present  in  the  proportion 
to  form  water.  The  term  is  not  strictly  appropriate  inasmuch  as 
there  are  bodies  such  as  acetic  acid,  lactic  acid  and  inosite  which 
have  H  and  O  present  in  the  proportion  to  form  water,  but  which 
are  not  carbohydrates,  and  there  are  also  true  carbohydrates  which 
do  not  have  H  and  O  present  in  this  proportion,  e.  g.,  rhamnose, 
C6H1205. 

Chemically  considered,  the  carbohydrates  are  aldehyde  or  ketone 
derivatives  of  complex  alcohols.  Treated  from  this  standpoint  the 
aldehyde  derivatives  are  spoken  of  as  aldoses,  and  the  ketone  deriva- 
tives are  spoken  of  as  ketoses.  The  carbohydrates  are  also  fre- 
quently named  according  to  the  number  of  oxygen  atoms  present  in 
the  molecule,  e.  g.,  trioses,  pentoses  and  hexoses. 

The  more  common  carbohydrates  may  be  classified  as  follows : 

I.  Monosaccharides. 

i.  Hexoses,  CGH12O0. 

(a)  Dextrose. 

(b)  Lsevulose. 

(c)  Galactose. 
2.  Pentoses,  C5H10O5. 

(a)  Arabinose. 

(b)  Xylose. 

(c)  Rhamnose  (Methyl-pentose),  C6H1205. 
II.  Disaccharides,  C12H22On. 

i.  Maltose. 

2.  Lactose. 

3.  Iso-Maltose. 

4.  Sucrose. 


22  PHYSIOLOGICAL    CHEMISTRY. 

III.  Trisaccharides,  C1SH3201G. 

i.  Raffinose. 

IV.  Polysaccharides,   (C6H10O5)x. 

i.   Starch  Group. 

(a)  Starch. 

(b)  Inulin. 

(c)  Glycogen. 

(d)  Lichenin. 

2.  Gum  and  Vegetable  Mucilage  Group. 

(a)  Dextrin. 

(b)  Vegetable  Gums. 

3.  Cellulose  Group. 

(a)  Cellulose. 

(b)  Hemi-Cellulose. 

Each  member  of  the  above  carbohydrate  classes,  except  the  mem- 
bers of  the  pentose  group,  may  be  supposed  to  contain  the  group 
C6H10O5  called  the  saccharide  group.  The  polysaccharides  consist 
of  this  group  alone  taken  a  large  number  of  times,  whereas  the 
disaccharides  may  be  supposed  to  contain  two  such  groups  plus  a 
molecule  of  water,  and  the  monosaccharides  to  contain  one  such 
group  plus  a  molecule  of  water.  Thus,  (C6H10O5)x  =  polysac- 
charide, (C6H10O5)2  -f-  H20  =  disaccharide,  C6H10O5  -f-  H20 
=  monosaccharide.  In  a  general  way  the  solubility  of  the  carbo- 
hydrates varies  with  the  number  of  saccharide  groups  present,  the 
substances  containing  the  largest  number  of  these  groups  being 
the  least  soluble.  This  means  simply  that,  as  a  class,  the  monosac- 
charides (hexoses)  are  the  most  soluble  and  the  polysaccharides 
(starches  and  cellulose)  are  the  least  soluble. 


MONOSACCHARIDES. 

Hexoses,  C6H1206. 

The  hexoses  are  monosaccharides  containing  six  oxygen  atoms 
in  the  molecule.  They  are  the  most  important  of  the  simple  sugars, 
and  two  of  the  principal  hexoses,  dextrose  and  lsevulose,  occur 
widely  distributed  in  plants  and  fruits.  Of  these  two  hexoses, 
dextrose  results  from  the  hydrolysis  of  starch  whereas  both  dextrose 
and  lsevulose  are  formed  in  the  hydrolysis  of  sucrose.  Galactose, 
which  with  dextrose  results  from  the  hydrolysis  of  lactose,  is  also 


CARBOHYDRATES.  23 

an  important  hexose.  These  three  hexoses  are  fermentable  by 
yeast,  and  yield  laevulinic  acid  upon  heating  with  dilute  mineral 
acids.  They  reduce  metallic  oxides  in  alkaline  solution,  are  optically 
active,  and  extremely  soluble.  With  phenylhydrazine  they  form 
characteristic  osazones. 

CH2OH 

DEXTROSE,  (CHOH)4. 

CHO 

Dextrose,  also  called  glucose  or  grape  sugar,  is  present  in  the 
blood  in  small  amount  and  may  also  occur  in  traces  in  normal  urine. 
After  the  ingestion  of  large  amounts  of  sucrose,  lactose  or  dex- 
trose, causing  the  assimilation  limit  to  be  exceeded,  an  alimentary 
glycosuria  may  arise.  In  diabetes  mellitus  very  large  amounts  of 
dextrose  are  excreted  in  the  urine.  The  following  structural  for- 
mula has  been  suggested  by  Victor  Meyer  for  d-dextrose : 

COH 
H  -  C  -  OH 

HO  -  C  -  H 

I 
H  -  C  -  OH 

H  -  C  -  OH 

CH2OH 

(For  further  discussion  of  dextrose  see  section  on  Hexoses, 
page  22.) 

Experiments  on  Dextrose. 

1.  Solubility. — Test  the  solubility  of  dextrose  in  the  "ordinary 
solvents"  and  in  alcohol.  (In  the  solubility  tests  throughout  the 
book  we  shall  designate  the  following-  solvents  as  the  "  ordinary 
solvents":  H20 ;  10  per  cent  NaCl;  0.5  per  cent  Na2COs ;  0.2  per 
cent  HC1;  concentrated  KOH;  concentrated  HC1.) 

2.  Molisch's  Reaction. — Place  approximately  5  c.c.  of  concen- 
trated H2S04  in  a  test-tube.  Incline  the  tube  and  slowly  pour  down 
the  inner  side  of  it  approximately  5  c.c.  of  the  sugar  solution  to 
which  2  drops  of  Molisch's  reagent  (a  15  per  cent  alcoholic  solution 


24  PHYSIOLOGICAL    CHEMISTRY. 

of  a-naphthol)  has  been  added,  so  that  the  sugar  solution  will  not 
mix  with  the  acid.  A  reddish-violet  zone  is  produced  at  the  point 
of  contact.     The  reaction  is  due  to  the  formation  of  furfurol, 

HC^CH 

HC      C-CHO, 

\/ 
0 

by  the  acid.  The  test  is  given  by  all  bodies  containing  a  carbohy- 
drate group  and  is  therefore  not  specific  and,  in  consequence,  of 
very  little  practical  importance. 

3.  Phenylhydrazine  Reaction. — Test  according  to  one  of  the 
following  methods:  (a)  To  a  small  amount  of  phenylhydrazine 
mixture,  furnished  by  the  instructor,1  add  5  c.c.  of  the  sugar  solu- 
tion, shake  well  and  heat  on  a  boiling  water-bath  for  one-half  to 
three-quarters  of  an  hour.  Allow  the  tube  to  cool  slowly  and 
examine  the  crystals  microscopically  (Plate  III,  opposite).  If  the 
solution  has  become  too  concentrated  in  the  boiling  process  it  will 
be  light-red  in  color  and  no  crystals  will  separate  until  it  is  diluted 
with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar 
giving  rise  to  an  osazone  of  a  definite  crystalline  form  which  is 
typical  for  that  sugar.  It  is  important  to  remember  in  this  connec- 
tion that  of  the  simple  sugars  of  interest  in  physiological  chemistry, 
dextrose  and  kevulose  yield  the  same  osazone.  Each  osazone  has  a 
definite  melting-point  and  as  a  further  and  more  accurate  means  of 
identification  it  may  be  recrystal'lized  and  identified  by  the  determi- 
nation of  its  melting-point  and  nitrogen  content.  The  reaction  tak- 
ing place  in  the  formation  of  phenyldextr osazone  is  as  follows : 

C6H1206  +  2(H2N-NH-C6H5)  = 

Dextrose.  Phenylhydrazine. 

C6H10O4(NNHC6H5)2  +  2H20  +  H2. 

Phenyldextrosazone. 

(b)  Place  5  c.c.  of  the  sugar  solution  in  a  test-tube,  add  1  c.c.  of 

1  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazine  hydro- 
chloride and  two  parts  of  sodium  acetate,  by  weight.  These  are  thoroughly 
mixed  in  a  mortar. 


PLATE    III. 


OSAZONES. 

Upper  form,  dextrosazone   or  lxvulosazone  ;   central   form,   maltosazone  ;   lower  form. 

lactosazone. 


CARBOHYDRATES. 


25 


the  phenylhydrazine-acetate  solution  furnished  by  the  instructor,1 
and  heat  on  a  boiling  water-bath  for  one-half  to  three-quarters  of 
an  hour.  Allow  the  liquid  to  cool  slowly  and  examine  the  crystals 
microscopically  (Plate  III,  opposite  p.  24). 

The  phenylhydrazine  test  has  been  so  modified  by  Cipollina  as 
to  be  of  use  as  a  rapid  clinical  test.  The  directions  for  this  test 
are  given  in  the  next  experiment. 

4.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  dextrose  solu- 
tion, 5  drops  of  phenylhydrazine  (the  base)  and  y2  c.c.  of  glacial 
acetic  acid  in  a  test-tube.  Heat  the  mixture  for  about  one  minute 
over  a  low  flame,  shaking  the  tube  continually  to  prevent  loss  of 
fluid  by  bumping.  Add  4-5  drops  of  sodium  hydroxide  (sp.  gr. 
1. 16),  being  certain  that  the  fluid  in  the  test-tube  remains  acid, 
heat  the  mixture  again  for  a  moment  and  then  cool  the  contents 
of  the  tube.  Ordinarily  the  crystals  form  at  once,  especially  if  the 
sugar  solution  possesses  a  low  specific  gravity.  If  they  do  not 
appear  immediately  allow  the  tube  to  stand  at  least  20  minutes 
before  deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  shown  in  Plate  III,  opposite  page  24. 

5.  Precipitation  by  Alcohol. — To  10  c.c.  of  95  per  cent  alcohol 
add  about  2  c.c.  of  dextrose  solution.  Compare  the  result  with 
that  obtained  under  Dextrin,  7,  page  49. 

Fig.   1. 


Dialyzixg  Apparatus  for  Students'  Use. 


6.  Iodine  Test.— Make  the  regular  iodine  test  as  given  under 
Starch,  5,  page  44,  and  keep  this  result  in  mind  for  comparison 
with  the  results  obtained  later  with  starch  and  with  dextrin. 

7.  Diffusibility  of  Dextrose. — Test  the  diffusibility  of  dextrose 

1  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case,  of 
glacial  acetic  acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the 
base). 


26  PHYSIOLOGICAL    CHEMISTRY. 

solution  through  animal  membrane,  or  parchment  paper,  making 
a  dialyzer  like  one  of  the  models  shown  in  Fig.  I,  p.  25. 

8.  Moore's  Test. — To  2-3  c.c.  of  sugar  solution  in  a  test-tube 
add  an  equal  volume  of  concentrated  KOH  or  NaOH,  and  boil. 
The  solution  darkens  and  finally  assumes  a  brown  color.  At  this 
point  the  odor  of  caramel  may  be  detected.  This  is  an  exceedingly 
crude  test  and  is  of  little  practical  value.  The  brown  color  is  due  to 
the  oxidation  of  the  dextrose  and  the  resulting  formation  of  the 
potassium  or  sodium  salts  of  certain  organic  acids  which  are  formed 
as  oxidation  products. 

9.  Reduction  Tests.  —  To  their  aldehyde  or  ketone  structure 
many  sugars  owe  the  property  of  readily  reducing  alkaline  solu- 
tions of  the  oxides  of  metals  like  copper,  bismuth  and  mercury; 
they  also  possess  the  property  of  reducing  ammoniacal  silver  solu- 
tions with  the  separation  of  metallic  silver.  Upon  this  property 
of  reduction  the  most  widely  used  tests  for  sugars  are  based.  When 
whitish-blue  cupric  hydroxide  in  suspension  in  an  alkaline  liquid 
is  heated  it  is  converted  into  insoluble  black  cupric  oxide,  but  if  a 
reducing  agent  like  certain  sugars  be  present  the  cupric  hydroxide 
is  reduced  to  insoluble  yellow  cuprous  hydroxide,  which  in  turn, 
on  further  heating,  may  be  converted  into  brownish-red  or  red 
cuprous  oxide.     These  changes  are  indicated  as  follows : 


OH 

/ 
Cu  s^Cu  =  0  +  H20. 

^v  Cupric  oxide 

^tt  (black). 

Cupric  hydroxide 
(whitish-blue). 


OH 

/ 

Cu 

\ 

OH 


2Cu-OH  +  H20  =  0. 


/ 

Cu 

\ 

OH 


f^TX  Cuprous  hydroxide 

(yellow). 


Cu-OH 

L- 


CARBOHYDRATES.  2J 

Cu 

X0  +  H20. 


OH 


/ 

Cu 


Cuprous  hydroxide  Cuprous  oxide 

(yellow).  (brownish-red). 

The  chemical  equations  here  discussed  are  exemplified  in  Trom- 
mer's  and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  sugar  solution  in  a  test- 
tube  add  one-half  its  volume  of  KOH  or  NaOH.  Mix  thor- 
oughly and  add,  drop  by  drop,  a  very  dilute  solution  of  cupric 
sulphate.  Continue  the  addition  until  there  is  a  slight  permanent 
precipitate  of  cupric  hydroxide  and  in  consequence  the  solution 
is  slightly  turbid.  Heat,  and  the  cupric  hydroxide  is  reduced  to 
yellow  cuprous  hydroxide  or  to  brownish-red  cuprous  oxide.  If 
the  solution  of  cupric  sulphate  used  is  too  strong  a  small  brownish- 
red  precipitate  produced  in  a  weak  sugar  solution  may  be  entirely 
masked.  On  the  other  hand,  particularly  in  testing  for  sugar  in 
the  urine,  if  too  little  cupric  sulphate  is  used  a  light-colored  pre- 
cipitate formed  by  uric  acid  and  purine  bases  may  obscure  the 
brownish-red  precipitate  of  cuprous  oxide.  The  action  of  KOH 
or  NaOH  in  the  presence  of  an  excess  of  sugar  and  insufficient 
copper  will  produce  a  brownish  color.  Phosphates  of  the  alkaline 
earths  may  also  be  precipitated  in  the  alkaline  solution  and  be 
mistaken  for  cuprous  hydroxide.  Trommer's  test  is  not  very  satis- 
factory. 

(b)  Fehling's  Test. — To  about  1  c.c.  of  Fehling's  solution1 
in  a  test-tube  add  about  4  c.c.  of  water,  and  boil.  This  is  done 
to  determine  whether  the  solution  will  of  itself  cause  the  forma- 
tion of  a  precipitate  of  brownish-red  cuprous  oxide.  If  such 
a  precipitate  forms,  the  Fehling's  solution  must  not  be 
used.  Add  sugar  solution  to  the  warm  Fehling's  solution  a  few 
drops  at  a  time  and  heat  the  mixture  after  each  addition.  The 
production  of  yellow  cuprous  hydroxide  or  brownish-red  cuprous 

1  Fehling's  solution  is  composed  of  two  definite  solutions— a  cupric  sulphate 
solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows : 

Cupric  sulphate  solution  =  34.65  grams  of  cupric  sulphate  dissolved  in  water 
and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  125  grams  of  potassium  hydroxide  and  173  grams 
■of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and 
mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to  prevent 
deterioration. 


28  PHYSIOLOGICAL    CHEMISTRY. 

oxide  indicates  that  reduction  has  taken  place.  The  yellow  pre- 
cipitate is  more  likely  to  occur  if  the  sugar  solution  is  added  rap- 
idly and  in  large  amount,  whereas  with  a  less  rapid  addition  of 
smaller  amounts  of  sugar  solution  the  brownish-red  precipitate  is 
generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even 
this  test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the 
urine.  Such  bodies  as  conjugate  glycuronates,  uric  acid,  nucieo- 
protein  and  homogentisic  acid  when  present  in  sufficient  amount 
may  produce  a  result  similar  to  that  produced  by  sugar.  Phos- 
phates of  the  alkaline  earths  may  be-  precipitated  by  the  alkali  of 
the  Fehling's  solution  and  in  appearance  may  be  mistaken  for 
cuprous  hydroxide.  Cupric  hydroxide  may  also  be  reduced  to 
cuprous  oxide  and  this  in  turn  be  dissolved  by  creatinine,  a  normal 
urinary  constituent.  This  will  give  the  urine  under  examination 
a  greenish  tinge  and  may  obscure  the  sugar  reaction  even  when  a 
considerable  amount  of  sugar  is  present. 

(c)  Benedict's  Modifications  of  Fehling's  Test. — First  Modifi- 
cation.— To  2  c.c.  of  Benedict's  solution1  in  a  test-tube  add  6  c.c. 
of  distilled  water  and  7-9  drops  (not  more)  of  the  solution  under 
examination.  Boil  the  mixture  vigorously  for  about  15-30  sec- 
onds and  permit  it  to  cool  to  room  temperature  spontaneously.  (If 
desired  this  process  may  be  repeated,  although  it  is  ordinarily  un- 
necessary.) If  sugar  is  present  in  the  solution  a  precipitate  will 
form  which  is  often  bluish-green  or  green  at  first,  especially  if  the 
percentage  of  sugar  is  low,  and  which  usually  becomes  yellowish 
upon  standing.  If  the  sugar  present  exceeds  0.06  per  cent  this 
precipitate  generally  forms  at  or  below  the  boiling  point,  whereas 
if  less  than  0.06  per  cent  of  sugar  is  present  the  precipitate  forms 
more  slowly  and  generally  only  after  the  solution  has  cooled. 

Benedict  claims  that,  whereas  the  original  Fehling  test  will  not 
serve  to  detect  sugar  when  present  in  a  concentration  of  less  than 
0.1  per  cent  that  the  above  modification  will  serve  to  detect  sugar 
when  present  in  as  small  quantity  as  0.015-0.02  per  cent. 

1  Benedict's  modified  Fehling  solution  consists  of  two  definite  solutions — a 
cupric  sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be  pre- 
pared as  follows : 

Cupric  sulphate  solution  =  34.65  grams  of  cupric  sulphate  dissolved  in  water 
and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  100  grams  of  anhydrous  sodium  carbonate  and 
173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles 
and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to  prevent 
deterioration. 


CARBOHYDRATES.  29 

The  modified  Fehling  solution  used  in  the  above  test  differs  from 
the  original  Fehling  solution  in  that  100  grams  of  sodium  car- 
bonate is  substituted  for  the  125  grams  of  potassium  hydroxide 
ordinarily  used,  thus  forming  a  Fehling  solution  which  is  consid- 
erably less  alkaline  than  the  original.  This  alteration  in  the  com- 
position of  the  Fehling  solution  is  of  advantage  in  the  detection 
of  sugar  in  the  urine  inasmuch  as  the  strong  alkalinity  of  the 
ordinary  Fehling'  solution  has  a  tendency,  when  the  reagent  is 
boiled  with  a  urine  containing  a  small  amount  of  dextrose,  to 
decompose  sufficient  of  the  sugar  to  render  the  detection  of  the 
remaining  portion  exceedingly  difficult  by  the  usual  technique. 
Benedict  claims  that  for  this  reason  the  use  of  his  modified  solu- 
tion permits  the  detection  of  much  smaller  amounts  of  sugar  than 
does  the  use  of  the  ordinary  Fehling  solution.  He  has  further 
modified  his  solution  for  use  in  the  quantitative  determination 
of  sugar  (see  Chapter  XXII). 

Second  Modification} — Very  recently  Benedict  has  further  modi- 
fied his  solution  and  has  succeeded  in  obtaining  one  which  does  not 
deteriorate  upon  long  standing.2  The  following  is  the  procedure 
for  the  detection  of  dextrose  in  solution :  To  five  cubic  centimeters 
of  the  reagent  in  a  test-tube  add  eight  (not  more)  drops  of  the 
solution  under  examination.  Boil  the  mixture  vigorously  for  from 
one  to  two  minutes  and  then  allow  the  fluid  to  cool  spontaneously. 
In  the  presence  of  dextrose  the  entire  body  of  the  solution  zvill  be 
filled  with  a  precipitate,  which  may  be  red,  yellow  or  green  in  color, 
depending  upon  the  amount  of  sugar  present.  If  no  dextrose  is 
present,  the  solution  will  remain  perfectly  clear.  (If  urine  is  being 
tested,  it  may  show  a  very  faint  turbidity,  due  to  precipitated 
urates.)      Even  very  small  quantities  of  dextrose   (o.  1  per  cent) 

1  Private  communication  from  Dr.  S.  R.  Benedict. 

2  Benedict's  new  solution  has  the  following  composition  : 

Cupric    sulphate    17.3  grams. 

Sodium   citrate    173.0  grams. 

Sodium   carbonate    (anhydrous) 100.0  grams. 

Distilled  water  to  make   1   liter. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600  c.c. 
of  water.  Pour  (through  a  folded  filter  paper  if  necessary)  into  a  glass  gradu- 
ate and  make  up  to  850  c.c.  Dissolve  the  cupric  sulphate  in  about  100  c.c.  of 
water  and  make  up  to  150  c.c.  Pour  the  carbonate-citrate  solution  into  a  large 
beaker  or  casserole  and  add  the  cupric  sulphate  solution  slowly,  with  constant 
stirring.  The  mixed  solution  is  ready  for  use  and  does  not  deteriorate  upon 
long  standing:. 


30  PHYSIOLOGICAL    CHEMISTRY. 

yield  precipitates  of  surprising  bulk  with  this  reagent,  and  the  posi- 
tive reaction  for  dextrose  is  the  filling  of  the  entire  body  of  the 
solution  with  a  precipitate,  so  that  the  solution  becomes  opaque. 
Since  amount  rather  than  color  of  the  precipitate  is  made  the  basis 
of  this  test,  it  may  be  applied,  even  for  the  detection  of  small  quan- 
tities of  dextrose,  as  readily  in  artificial  light  as  in  daylight. 

(d)  Boettger's  Test. — To  5  c.c.  of  sugar  solution  in  a  test-tube 
add  1  c.c.  of  KOH  or  NaOH  and  a  very  small  amount  of  bismuth 
subnitrate,  and  boil.  The  solution  will  gradually  darken  and 
finally  assume  a  black  color  due  to  reduced  bismuth.  If  the  test 
is  made  on  urine  containing  albumin  this  must  be  removed,  by 
boiling  and  filtering,  before  applying  the  test,  since  with  albumin 
a  similar  change  of  color  is  produced   (bismuth  sulphide). 

(e)  Nylander's  Test  (Almen's  Test). — To  5  c.c.  of  sugar  solu- 
tion in  a  test-tube  add  one-tenth  its  volume  of  Nylander's  reagent1 
and  heat  for  five  minutes  in  a  boiling  water-bath.2  The  solution 
will  darken  if  reducing  sugar  is  present  and  upon  standing  for  a 
few  moments  a  black  color  will  appear.  This  color  is  due  to  the 
precipitation  of  bismuth.  If  the  test  is  made  on  urine  containing 
albumin  this  must  be  removed,  by  boiling  and  filtering,  before 
applying  the  test,  since  with  albumin  a  similar  change  of  color  is 
produced.  Dextrose  when  present  to  the  extent  of  0.08  per  cent, 
may  be  detected  by  this  reaction.  It  is  claimed  by  Bechold  that 
Nylander's  and  Boettger's  tests  give  a  negative  reaction  with 
solutions  containing  sugar  when  mercuric  chloride  or  chloroform 
is  present.  Other  observers  have  failed  to  verify  the  inhibitory 
action  of  mercuric  chloride  and  have  shown  that  the  inhibitory  in- 
fluence of  chloroform  may  be  overcome  by  raising  the  tempera- 
ture of  the  urine  to  the  boiling-point  for  a  period  of  five  minutes 
previous  to  making  the  test.  Urines  rich  in  indican,  uroerythrin 
or  hcemaio  porphyrin,  as  well  as  urines  excreted  after  the  inges- 
tion of  large  amounts  of  certain  medicinal  substances,  may  give 
a  darkening  of  Nylander's  reagent  similar  to  that  of  a  true  sugar 
reaction. 

According  to  Rustin  and  Otto  the  addition  of  PtCl4  increases 
the   delicacy  of  Nylander's  reaction.     They  claim  that  this   pro- 

'  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  subnitrate 
and  a  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium  hydroxide 
solution.     The  reagent  is  then  cooled  and  filtered. 

2  Hammarsten  suggests  that  the  mixture  should  be  boiled  2-5  minutes  (accord- 
ing to  the  sugar  content)  over  a  free  flame  and  the  tube  then  permitted  to  stand 
K  minutes  before  drawing  conclusions. 


CARBOHYDRATES. 


31 


cedure  causes  the  sugar  to  be  converted  quantitatively.  No  quan- 
titative method  has  yet  been  devised,  however,  based  upon  this 
principle. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to  the  fol- 


FlG.    2. 


lowing  reactions : 

(a)  Bi(OH)2NO,  +  KOH  =  Bi(OH)8  +  KN03. 

(b)  2Bi(OH)3  -  30  =  Bi2  +  3H20. 

10.  Fermentation  Test. — "  Rub  up  "  in  a  mortar  about  20 
c.c.  of  the  sugar  solution  with  a  small  piece  of  compressed  yeast. 
Transfer  the  mixture  to  a  saccharometer 
( shown  in  Fig.  2)  and  stand  it  aside  in  a 
warm  place  for  about  twelve  hours.  If 
the  sugar  is  fermentable,  alcoholic  fermen- 
tation will  occur  and  carbon  dioxide  will 
collect  as  a  gas  in  the  upper  portion  of  the 
tube.  On  the  completion  of  fermentation 
introduce  a  little  potassium  hydroxide  solu- 
tion into  the  graduated  portion  by  means 
of  a  bent  pipette,  place  the  thumb  tightly 
over  the  opening  in  the  apparatus  and  in- 
vert the  saccharometer.     Explain  the  result. 

11.  Barfoed's  Test. — Place  .about  5  c.c. 
of  Barfoed's  solution1  in  a  test-tube  and 
heat  to  boiling.  Add  dextrose  solution 
slowly,  a  few  drops  at  a  time,  heating  after 
each  addition.  Reduction  is  indicated  by 
the  formation  of  a  red  precipitate.  If 
the  precipitate  does  not  form  upon  continued 
boiling  allow  the  tube  to  stand  a  few  minutes  and  examine.  Sodium 
chloride  interferes  with  the  reaction  (Welker). 

Barfoed's  test  is  not  a  specific  test  for  dextrose  as  is  frequently 
stated,  but  simply  serves  to  detect  monosaccharides.  Disac- 
charides  will  also  respond  to  the  test,  according  to  Hinkel  and 
Sherman,  if  the  sugar  solution  is  boiled  sufficiently  long,  in  contact 
with  the  reagent,  to  hydrolyze  the  disaccharide  through  the  action 
of  the  acetic  acid  present  in  the  Barfoed's  solution. 

12.  Formation  of  Caramel. — Gently  heat  a  small  amount  of 

1  Barfoed's  solution  is  prepared  as  follows :  Dissolve  4.5  grams  of  neutral, 
crystallized  cupric  acetate  in  100  c.c.  of  water  and  add  0.12  c.c.  of  50  per  cent 
acetic  acid. 


Einhorx    Saccharometer. 


32  PHYSIOLOGICAL    CHEMISTRY. 

pulverized  dextrose  in  a  test-tube.  After  the  sugar  has  melted  and 
turned  brown,  allow  the  tube  to  cool,  add  water  and  warm.  The 
coloring'  matter  produced  is  known  as  caramel. 

13.  Demonstration  of  Optical  Activity. — A  demonstration  of 
the  use  of  the  polariscope,  by  the  instructor,  each  student  being, 
required  to  take  readings  and  compute  the  "  specific  rotation." 

Use    of    the    Polariscope. 

For  a  detailed  description  of  the  different  forms  of  polariscopes, 
the  method  of  manipulation  and  the  principles  involved  the  student 
is  referred  to  any  standard  text-book  of  physics.  A  brief  descrip- 
tion follows :  An  ordinary  ray  of  light  vibrates  in  every  direction. 
If  such  a  ray  is  caused  to  pass  through  a  "  polarizing "  Nicol 
prism  it  is  resolved  into  two  rays,  one  of  which  vibrates  in  every 
direction  as  before  and  a  second  ray  which  vibrates  in  one  plane 
only.  This  latter  ray  is  said  to  be  polarized.  Many  organic  sub- 
stances (sugars,  proteins,  etc.)  have  the  power  of  twisting  or  rotat- 
ing this  plane  of  polarized  light,  the  extent  to  which  the  plane  is 
rotated  depending  upon  the  number  of  molecules  which  the  polar- 
ized light  passes.  Substances  which  possess  this  power  are  said  to 
be  "  optically  active."  The  specific  rotation  of  a  substance  is  the 
rotation  expressed  in  degrees  which  is  afforded  by  one  gram  of 
substance  dissolved  in  1  c.c.  of  water  in  a  tube  one  decimeter  in 
length.  The  specific  rotation,  (a)v,  may  be  calculated  by  means 
of  the   following   formula, 

in  which 

d  =  sodium  light. 

a  ==  observed   rotation   in   degrees. 

p  =  grams  of  substance  dissolved  in   1   c.c.  of  liquid. 
/  =  length   of   the   tube   in   decimeters. 
If  the  specific  rotation  has  been  determined  and  it  is  desired  to 
ascertain  the  per  cent  of  the  substance  in  solution,  this  may  be 
obtained  by  the  use  of  the  following  formula, 


P  = 


(«V 


The  value  of  p  multiplied  by   100  will  be  the  percentage  of  the 
substance  in  solution. 


CARBOHYDKATKS. 


33 


An  instrument  by  means  of  which  the  extent  of  the  rotation  may 
be  determined  is  called  a  polariscope  or  polarimeter.  Such  an  in- 
strument designed  especially  for  the  examination  of  sugar  solutions 
is  termed  a  saccharimeter  or  polarizing  saccharimeter.     The  form  of 


Fig.  3. 


One  Form  of  Laurent  Polariscope. 


B,  Microscope  for  reading  the  scale ;  C,  a  vernier ;  E,  position  of  the  analyzing  Nicol 
prism ;  H,  polarizing  Nicol  prism  in  the  tube  below  this  point. 

polariscope  shown  in  Fig.  3,  above,  consists  essentially  of  a  long 
barrel  provided  with  a  Nicol  prism  at  either  end  (Fig.  4,  below). 
The  solution  under  examination  is  contained  in  a  tube  which  is 
placed  between  these  two  prisms.  At  the  front  end  of  the  instru- 
ment is  an  adjusting  eye-piece  for  focusing  and  a  large  recording 


Fig.  4. 


HO 


^> 


Diagrammatic  Representation  of  the  Course  of  the  Light  through  the  Laurent 
Polariscope.     (The  direction  is  reversed  from  that  of  Fig.   3,  above.) 

a,  Bichromate  plate  to  purify  the  light ;  b,  the  polarizing  Nicol  prism  ;  c,  a  thin 
quartz  plate  covering  one-half  the  field  and  essential  in  producing  a  second  polarized 
plane  ;  d,  tube  to  contain  the  liquid  under  examination  ;  e,  the  analyzing  Nicol  prism  ; 
f  and  g,  ocular  lenses. 


disc  which  registers  in  degrees  and  fractions  of  a  degree.  The  light 
is  admitted  into  the  far  end  of  the  instrument  and  is  polarizad  by 
passing  through  a  Nicol  prism.  This  polarized  ray  then  traverses 
the  column  of  liquid  within  the  tube  mentioned  above  and  if  the  sub- 

4 


34 


PHYSIOLOGICAL    CHEMISTRY. 


stance  is  optically  active  the  plane  of  the  polarized  ray  is  rotated  to 
the  right  or  left.  Bodies  rotating  the  ray  to  the  right  are  called 
dextro-rotatory  and  those  rotating  it  to  the  left  Icevo-rotatory. 

Within  the  apparatus  is  a  disc  which  is  so  arranged  as  to  be 
without  lines  and  uniformly  light  at  zero.     Upon  placing  the  opti- 


Fig.  5. 


Polariscope    (Schmidt  and   Hansch    Model). 


cally  active  substance  in  position,  however,  the  plane  of  polarized 
light  is  rotated  or  turned  and  it  is  necessary  to  rotate  the  disc 
through  a  certain  number  of  degrees  in  order  to  secure  the  normal 
conditions,  i.  e.,  "  without  lines  and  uniformly  light."  The  differ- 
ence between  this  reading  and  the  zero  is  a  or  the  observed  rotation 
in  degrees. 

Polarizing  saccharimeters  are  also  constructed  by  which  the  per- 
centage of  sugar  in  solution  is  determined  by  making  an  observa- 
tion and  multiplying  the  value  of  each  division  on  a  horizontal  slid- 
ing scale  by  the  value  of  the  division  expressed  in  terms  of  dex- 
trose.    This  factor  may  vary  according  to  the  instrument. 


CARBOHYDRATES.  35 

CH2OH 

LJEVULOSE,  (CHOH)3. 

CO 
CHoOH 


As  already  stated,  lsevulose,  sometimes  called  fructose  or  fruit 
sugar,  occurs  widely  disseminated  throughout  the  plant  kingdom 
in  company  with  dextrose.  Its  reducing  power  is  somewhat  weaker 
than  that  of  dextrose.  Lsevulose  does  not  ordinarily  occur  in  the 
urine  in  diabetes  mellitus,  but  has  been  found  in  exceptional  cases. 
With  phenylhydrazine  it  forms  the  same  osazone  as  dextrose.  With 
methylphenylhydrazine,  lsevulose  forms  a  characteristic  methyl- 
phenyllsevulosazone. 

(For  a  further  discussion  of  lsevulose  see  the  section  on  Hexoses, 

p.    22.) 

Experiments  on  L^vulose. 

I— ii.  Repeat  these  experiments  as  given  under  Dextrose,  pages 

23-3I- 

12.  Seliwanoff's  Reaction. — To  5  c.c.  of  Seliwanoff's  reagent1 

in  a  test-tube  add  a  few  drops  of  a  lsevulose  solution  and  heat  the 
mixture  to  boiling.  A  positive  reaction  is  indicated  by  the  produc- 
tion of  a  red  color  and  the  separation  of  a  red  precipitate.  The 
latter  may  be  dissolved  in  alcohol  to  which  it  will  impart  a  striking 
red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained 
with  solutions  of  dextrose  or  maltose. 

13.  Borchardt's  Reaction. — To  about  5  c.c.  of  a  solution  of 
lsevulose  in  a  test-tube  add  an  equal  volume  of  25  per  cent  hydro- 
chloric acid  and  a  few  crystals  of  resorcin.  Heat  to  boiling  and 
after  the  production  of  a  red  color,  cool  the  tube  under  running 
water  and  transfer  to  an  evaporating  dish  or  beaker.  Make  the 
mixture  slightly  alkaline  with  solid  potassium  hydroxide,  return  it 
to  a  test  tube,  add  2-3  c.c.  of  acetic  ether  and  shake  the  tube  vig- 
orously. In  the  presence  of  lsevulose,  the  acetic  ether  is  colored 
yellow.     (For  further  discussion  of  the  test  see  Chapter  XIX.) 

14.  Formation  of  Methylphenyllaevulosazone. — To  a  solution 

1  Seliwanoff's  reagent  may  be  prepared  by  dissolving  0.05  gram  of  resorcin 
in  100  c.c.  of  dilute   (1:2)   hydrochloric  acid. 


36  PHYSIOLOGICAL    CHEMISTRY. 

of  1.8  gram  of  lsevulose  in  10  c.c.  of  water  add  4  grams1  of  methyl- 
phenylhydrazine  and  enough  alcohol  to  clarify  the  solution.  Intro- 
duce 4  c.c.  of  50  per  cent  acetic  acid  and  heat  the  mixture  for  5-10 
minutes  on  a  boiling  water-bath.2  On  standing  15  minutes  at  room 
temperature,  crystallization  begins  and  is  complete  in  two  hours. 
By  scratching  the  sides  of  the  flask  or  by  inoculation,  the  solution 
quickly  congeals  to  form  a  thick  paste  of  reddish  yellow  silky 
needles.  These  are  the  crystals  of  methylphenyllcevulosazone. 
They  may  be  recrystallized  from  hot  95  per  cent  alcohol  and  melt  at 

153°  C 

CH2OH 

GALACTOSE,  (CHOH)4. 

CHO 

Galactose  occurs  with  dextrose  as  one  of  the  products  of  the 
hydrolysis  of  lactose.  It  is  dextro-rotatory,  forms  an  osazone  with 
phenylhydrazine  and  ferments  slowly  with  yeast.  Upon  oxida- 
tion with  nitric  acid  galactose  yields  mucic  acid,  thus  differentiat- 
ing this  monosaccharide  from  dextrose  and  lsevulose.  Lactose  also 
yields  mucic  acid  under  these  conditions.  The  mucic  acid  test  may 
be  used  in  urine  examination  to  differentiate  lactose  and  galactose 
from  other  reducing  sugars. 

Experiments    on    Galactose. 

1.  Tollens'  Reaction. — To  equal  volumes  of  galactose  solu- 
tion and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucin, 
and  heat  the  mixture  on  a  boiling  water-bath.  Galactose,  pentose 
and  glycuronic  acid  will  be  indicated  by  the  appearance  of  a  red 
color.  Galactose  may  be  differentiated  from  the  two  latter  sub- 
stances in  that  its  solutions  exhibit  no  absorption  bands  upon  spec- 
Iroscopical  examinations. 

2.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing 
galactose  with  20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and 
evaporate  the  mixture  in  a  broad,  shallow  glass  vessel  on  a  boiling 
water-bath  until  the  volume  of  the  mixture  has  been  reduced  to 
about  20  c.c.  At  this  point  the  fluid  should  be  clear,  and  a  .fine 
white  precipitate  of  mucic  acid  should  form.  If  the  percentage  of 
galactose  present  is  low  it  may  be  necessary  to  cool  the  solution 

1  3.66  grams  if  absolutely  pure. 

2  Longer  heating  is  to  be  avoided. 


CARBOHYDRATES.  37 

and  permit  it  to  stand  for  some  time  before  the  precipitate  will  form. 
It  is  impossible  to  differentiate  between  galactose  and  lactose  by 
this  test,  but  the  reaction  serves  to  differentiate  these  two  sugars 
from  all  other  reducing  sugars.  Differentiate  lactose  from  galac- 
tose by  means  of  Barfoed's  test  (p.  31). 

3.  Phenylhydrazine  Reaction. — Make  the  test  according  to 
directions  given  under  Dextrose,  3  or  4,  pages  24  and  25. 

Pentoses,    C5H10O5. 

In  plants  and  more  particularly  in  certain  gums,  very  complex 
carbohydrates,  called  pentosans,  occur.  These  pentosans  through 
hydrolysis  by  acids  may  be  transformed  into  pentoses.  Pentoses 
do  not  ordinarily  occur  in  the  animal  organism,  but  have  been 
found  in  the  urine  of  morphine  habitues  and  others,  their  occur- 
rence sometimes  being  a  persistent  condition  without  known  cause. 
They  are  non- fermentable,  have  strong  reducing  power  and  form 
osazones  with  phenylhydrazine.  Pentoses  are  an  important  constitu- 
ent of  the  dietary  of  herbivorous  animals.  Glycogen  is  said  to  be 
formed  after  the  ingestion  of  these  sugars  containing  five  oxygen 
atoms.  This,  however,  has  not  been  conclusively  proven.  On 
distillation  with  strong  hydrochloric  acid  pentoses  and  pentosans 
yield  furfurol,  which  can  be  detected  by  its  characteristic  red  reac- 
tion with  aniline-acetate  paper. 

CH2OH 

ARABINOSE,    (CHOH)3. 

CHO 

Arabinose  is  one  of  the  most  important  of  the  pentoses.  The 
/-arabinose  may  be  obtained  from  gum  arabic,  plum  or  cherry  gum 
by  boiling  for  several  hours  with  1-2  per  cent  sulphuric  acid. 
This  pentose  is  dextrorotatory,  forms  an  osazone  and  has  reducing 
power.  The  i-arabinose  has  been  isolated  from  the  urine  and 
yields  an  osazone  which  melts  at  166-1680  C. 

Experiments  on  Arabinose. 

1.  Tollens'  Reaction. — To  equal  volumes  of  arabinose  solu- 
tion and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucin 
and  heat  the  mixture  on  a  boiling  water-bath.  Galactose,  pentose 
or  glycuronic  acid  will  lie  indicated  by  the  appearance  of  a  red 


38  PHYSIOLOGICAL    CHEMISTRY. 

color.  To  differentiate  between  these  bodies  make  a  spectroscopic 
examination  and  look  for  the  absorption  band  between  D  and  E 
given  by  pentoses  and  glycuronic  acid.  Differentiate  between  the 
two  latter  bodies  by  the  melting-points  of  their  osazones. 

Compare  the  reaction  with  that  obtained  with  galactose  (page  36) . 

2.  Orcin  Test. — Repeat  1,  using  orcin  instead  of  phloroglucin. 
A  succession  of  colors  from  red  through  reddish-blue  to  green 
is  produced.  A  green  precipitate  is  formed  which  is  soluble  in 
amyl  alcohol  and  has  absorption  bands  between  C  and  D. 

3.  Phenylhydrazine  Reaction. — Make  this  test  on  the  arabinose 
solution  according  to  directions  given  under  Dextrose,  3  or  4, 
pages  24  and  25. 

CH2OH 

xylose,  (CHOH)3. 

I 
CHO 

Xylose,  or  wood  sugar,  is  obtained  by  boiling  wood  gums  with 
dilute  acids  as  explained  under  Arabinose,  page  37.  It  is  dextro- 
rotatory and  forms  an  osazone. 

Experiments  on  Xylose. 
1-3.  Same  as  for  arabinose  (see  page  37). 

RHAMNOSE,  C6H1205. 

Rhamnose  or  methyl-pentose  is  an  example  of  a  true  carbohydrate 
which  does  not  have  the  H  and  O  atoms  present  in  the  proportion 
to  form  water.  Its  formula  is  C0H12O5.  It  has  been  found  that 
rhamnose  when  ingested  by  rabbits  or  hens  has  a  positive  influence 
upon  the  formation  of  glycogen  in  those  organisms. 


DISACCHARIDES,  C12H22On. 

The  disaccharides  as  a  class  may  be  divided  into  two  rather  dis- 
tinct groups.  The  first  group  would  include  those  disaccharides 
which  are  found  in  nature  as  such,  e.  g.,  sucrose  and  lactose 
and  the  second  group  would  include  those  disaccharides  formed  in 
the  hydrolysis  of  more  complex  carbohydrates,  e.  g.,  maltose,  and 
iso-maltose. 


CARBOHYDRATES.  39 

The  disaccharides  have  the  general  formula  C]2H22On,  to  which, 
in  the  process  of  hydrolysis,  a  molecule  of  water  is  added  causing 
the  single  disaccharide  molecule  to  split  into  two  monosaccharide 
(hexose)  molecules.  The  products  of  the  hydrolysis  of  the  more 
common  disaccharides  are  as  follows : 

Maltose  —  dextrose  -J-  dextrose. 
Lactose  =  dextrose  -\-  galactose. 
Sucrose  =  dextrose  -\-  lsevulose. 

All  of  the  more  common  disaccharides  except  sucrose  have 
the  power  of  reducing  certain  metallic  oxides  in  alkaline  solution, 
notably  those  of  copper  and  bismuth.  This  reducing  power  is  due 
to  the  presence  of  the  aldehyde  group  ( — CHO)  in  the  sugar 
molecule. 

MALTOSE,  CjaHaaOn. 

Maltose  or  malt  sugar  is  formed  in  the  hydrolysis  of  starch 
through  the  action  of  an  enzyme,  vegetable  amylase  (diastase) ,  con- 
tained in  sprouting  barley  or  malt.  Certain  enzymes  in  the  saliva 
and  in  the  pancreatic  juice  may  also  cause  a  similar  hydrolysis. 
Maltose  is  also  an  intermediate  product  of  the  action  of  dilute  mineral 
acids  upon  starch.  It  is  strongly  dextro-rotatory,  reduces  metallic 
oxides  in  alkaline  solution  and  is  fermentable  by  yeast  after  being 
inverted  (see  Polysaccharides,  page  43)  by  the  enzyme  maltase 
of  the  yeast.  In  common  with  the  other  disaccharides,  maltose  may 
be  hydrolyzed  with  the  formation  of  two  molecules  of  monosac- 
charide. In  this  instance  the  products  are  two  molecules  of  dex- 
trose. With  phenylhydrazine  maltose  forms  an  osazone,  maltosa- 
zone.  The  following  formula  represents  the  probable  structure  of 
maltose : 

CH2OH        CHO 

CHOH         CHOH 


4q  physiological  chemistry. 

Experiments  on  Maltose. 

i-ii.  Repeat  these  experiments  as  given  under  Dextrose,  pages 

23-3I- 

ISO-MALTOSE,  C^H^On. 

Iso-maltose,  an  isomeric  form  of  maltose,  is  formed,  along  with 
maltose,  by  the  action  of  diastase  upon  starch  paste,  and  also  by 
the  action  of  hydrochloric  acid  upon  dextrose.  It  also  occurs  with 
maltose  as  one  of  the  products  of  salivary  digestion.  It  is  dextro- 
rotatory and  with  phenylhydrazine  gives  an  osazone  which  is  char- 
acteristic. Iso-maltose  is  very  soluble  and  reduces  the  oxides  of 
bismuth  and  copper  in  alkaline  solution.  Pure  iso-maltose  is  prob- 
ably only  slightly  fermentable. 

LACTOSE,  C^H^On. 

Lactose  or  milk  sugar  occurs  ordinarily  only  in  milk,  but  has 
often  been  found  in  the  urine  of  women  during  pregnancy  and 
lactation.  It  may  also  occur  in  the  urine  of  normal  persons  after 
the  ingestion  of  unusually  large  amounts  of  lactose  in  the  food. 
It  has  a  strong  reducing  power,  is  dextro-rotatory  and  forms  an 
osazone  with  phenylhydrazine.  Upon  hydrolysis  lactose  yields  one 
molecule  of  dextrose  and  one  molecule  of  galactose. 

In  the  souring  of  milk  the  bacterium  lactis  and  certain  other 
micro-organisms  bring  about  lactic  acid  fermentation  by  transform- 
ing the  lactose  of  the  milk  into  lactic  acid, 

H      OH 

H  -  C  -  C  -  COOH, 

I         I 
H      H 

and  alcohol.  This  same  reaction  may  occur  in  the  alimentary  canal 
as  the  result  of  the  action  of  putrefactive  bacteria.  In  the  prepara- 
tion of  kephyr  and  koumyss  the  lactose  of  the  milk  undergoes  alco- 
holic fermentation,  through  the  action  of  ferments  other  than  yeast, 
and  at  the  same  time  lactic  acid  is  produced.  Lactose  and  galactose 
yield  mucic  acid  on  oxidation  with  nitric  acid.  This  fact  is  made 
use  of  in  urine  analysis  to  facilitate  the  differentiation  of  these 
sugars  from  other  reducing  sugars. 

Lactose  is  not  fermentable  by  pure  yeast. 


carbohydrates.  41 

Experiments  on  Lactose. 
i— i  i.     Repeat  these  experiments  as  given  under  Dextrose,  pages 

23-31. 

12.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing 
lactose  with  20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and 
evaporate  the  mixture  in  a  broad,  shallow,  glass  vessel  on  a  boiling 
water-bath,  until  the  volume  of  the  mixture  has  been  reduced  to 
about  20  c.c.  At  this  point  the  fluid  should  be  clear,  and  a  fine  white 
precipitate  of  mucic  acid  should  form.  If  the  percentage  of  lactose 
present  is  low  it  may  be  necessary  to  cool  the  solution  and  permit 
it  to  stand  for  some  time  before  the  precipitate  will  appear.  It  is 
impossible  to  differentiate  between  lactose  and  galactose  by  this  test, 
but  the  reaction  serves  to  differentiate  these  two  sugars  from  all 
other  reducing  sugars. 

Differentiate  lactose  from  galactose  by  means  of  Barfoed's  test, 
page  31. 

SUCROSE,   CioHooOn. 

Sucrose,  also  called  saccharose  or  cane  sugar,  is  one  of  the  most 
important  of  the  sugars  and  occurs  very  extensively  distributed  in 
plants,  particularly  in  the  sugar  cane,  sugar  beet,  sugar  millet  and 
in  certain  palms  and  maples. 

Sucrose  is  dextro-rotatory  and  upon  hydrolysis,  as  before  men- 
tioned, the  molecule  of  sucrose  takes  on  a  molecule  of  water  and 
breaks  down  into  two  molecules  of  monosaccharide.  The  mono- 
saccharides formed  in  this  instance  are  dextrose  and  lsevulose.  This 
is  the  reaction : 

G12R22Oxl  +  H20  =  C6H1206  -f-  C6H1206. 

Sucrose.  Dextrose.  Lsevulose. 

This  process  is  called  inversion  and  may  be  produced  by  bacteria, 
enzymes  and  certain  weak  acids.  After  this  inversion  the  previously 
strongly  dextro-rotatory  solution  becomes  laevo-rotatory.  This  is 
due  to  the  fact  that  the  lsevulose  molecule  is  more  strongly  lawo- 
rotatory  than  the  dextrose  molecule  is  dextro-rotatory.  The  product 
of  this  inversion  is  called  invert  sugar. 

Sucrose  does  not  reduce  metallic  oxides  in  alkaline  solution 
and  forms  no  osazone  with  phenylhydrazine.  It  is  not  fermentable 
directly  by  yeast,  but  must  first  be  inverted  by  the  enzyme  sitcrase 
(invertase  or  inverting  contained  in  the  yeast.  The  probable  struc- 
ture  of    sucrose   may   be   represented   by   the    following    formula. 


42 


PHYSIOLOGICAL    CHEMISTRY. 


Note  the  absence  of  any" true  sugar  group  or  free  ketone  or  alde- 
hyde group. 

CH2OH 

CHOH 

CHO 


-0     CH2OH 


H 


Sucrose. 


Fig.  6. 


Experiments  on  Sucrose. 

i-ii.  Repeat  these  experiments  according  to  the  directions  given 
under  Dextrose,  pages  23-31. 

12.  Inversion  of  Sucrose. — To  25  c.c.  of  sucrose  solution  in 
a  beaker  add  5  drops  of  concentrated  HC1  and  boil  one  minute. 
Cool  the  solution,  render  alkaline  with  solid  KOH  and  upon  the 
resulting  fluid  repeat  experiments  3  (or  4)  and  9  as  given  under 
Dextrose,  pages  24-26.     Explain  the  results. 

13.  Production  of  Alcohol  by  Fermentation.  —  Prepare  a 
strong  (10-20  per  cent)   solution  of  sucrose,  add  a  small  amount 

of  egg  albumin  or  commercial  peptone 
and  introduce  the  mixture  into  a  bottle 
of  appropriate  size.  Add  yeast,  and  b)^ 
means  of  a  bent  tube  inserted  through  a 
stopper  into  the  neck  of  the  bottle,  con- 
duct the  escaping  gas  into  water.  As 
fermentation  proceeds  readily  in  a  warm 
place  the  escaping  gas  may  be  collected 
in  a  eudiometer  tube  and  examined. 
When  the  activity  of  the  yeast  has  prac- 
tically ceased,  filter  the  contents  of  the 
bottle  into  a  flask  and  distil  the  mixture. 
Catch  the  first  portion  of  the  distillate  separately  and  test  for  alco- 
hol by  one  of  the  following  reactions : 

(a)  Iodoform  Test. — Render  2-3  c.c.  of  the  distillate  alkaline 
with  potassium  hydroxide  solution  and  add  a  few  drops  of  iodine 


Iodoform.     (Autenrietk.) 


CARBOHYDRATES.  43 

solution.  Heat  gently  and  note  the  formation  of  iodoform  crystals. 
Examine  these  crystals  under  the  microscope  and  compare  them 
with  those  in  Fig.  6,  p.  42. 

(b)  Aldehyde  Test.— Place  5  c.c.  of  the  distillate  in  a  test-tube, 
add  a  few  drops  of  potassium  dichromate  solution,  K2Cr207,  and 
render  it  acid  with  dilute  sulphuric  acid.  Boil  the  acid  solution  and 
note  the  odor  of  aldehyde. 


TRISACCHARIDES,  C1SH32016. 

RAFFINOSE. 

This  trisaccharide,  also  called  melitose  or  melitriose,  occurs  in 
cotton  seed,  Australian  manna  and  in  the  molasses  from  the  prepara- 
tion of  beet  sugar.  It  is  dextro-rotatory,  does  not  reduce  Fehling's 
solution  and  is  only  partially  fermentable  by  yeast. 

Raffinose  may  be  hydrolyzed  by  weak  acids  the  same  as  the  poly- 
saccharides are  hydrolyzed,  the  products  being  lsevulose  and  meli- 
biose;  further  hydrolysis  of  the  melibiose  yields  dextrose  and 
galactose. 

POLYSACCHARIDES,   (C6H10O5)x. 

In  general  the  polysaccharides  are  amorphous  bodies,  a  few,  how- 
ever, are  crystallizable.  Through  the  action  of  certain  enzymes 
or  weak  acids  the  polysaccharides  may  be  hydrolyzed  with  the  for- 
mation of  monosaccharides.  As  a  class  the  polysaccharides  are 
quite  insoluble  and  are  non-fermentable  until  inverted.  By  inversion 
is  meant  the  hydrolysis  of  disaccharide  or  polysaccharide  sugars 
to  form  monosaccharides,  as  indicated  in  the  following  equations : 

(a)  C12H22On  +  H2O  =  2(CfiH12O0). 

(b)  CflH10OB  +  H2O  =  C6H"12Oe. 

STARCH,  (C6H1005)x. 

Starch  is  widely  distributed  throughout  the  vegetable  kingdom, 
occurring  in  grains,  fruits  and  tubers.  It  occurs  in  granular  form, 
the  microscopical  appearance  being  typical  for  each  individual 
starch.  The  granules,  which  differ  in  size  according  to  the  source, 
are  composed  of  alternating  concentric  rings  of  granulose  and  cel- 
lulose. Ordinary  starch  is  insoluble  in  cold  water,  but  if  boiled 
with  water  the  cell  walls  are  ruptured  and  starch  paste  results.  In 
general  starch  gives  a  blue  color  with  iodine. 


44  PHYSIOLOGICAL    CHEMISTRY. 

Starch  is  acted  upon  by  amylases,  e.  g.,  salivary  amylase  (ptyalin) 
and  pancreatic  amylase  (amylopsin) ,  with  the  formation  of  soluble 
starch,  erythro-dextrin,  achroo-dextrins ,  maltose,  iso-maltose  and 
dextrose  (see  Salivary  Digestion,  page  54).  Maltose  is  the 
principal  end-product  of  this  enzyme  action.  Upon  boiling  a  starch 
solution  with  a  dilute  mineral  acid  a  series  of  similar  bodies 
is  formed,  but  under  these  conditions  dextrose  is  the  principal 
end-product. 

Experiments  on  Starch. 

1.  Preparation  of  Potato  Starch. — Pare  a  raw  potato,  com- 
minute it  upon  a  fine  grater,  mix  with  water,  and  "whip  up"  the 
pulped  material  vigorously  before  straining  it  through  cheese  cloth 
or  gauze  to  remove  the  coarse  particles.  The  starch  rapidly  settles 
to  the  bottom  and  can  be  washed  by  repeated  decantation.  Allow 
the  compact  mass  of  starch  to  drain  thoroughly  and  spread  it  out 
on  a  watch  glass  to  dry  in  the  air.  If  so  desired  this  preparation 
may  be  used  in  the  experiments  which  follow. 

2.  Microscopical  Examination. — Examine  microscopically  the 
granules  of  the  various  starches  submitted  and  compare  them  with 
those  shown  in  Figs.  7-17,  page  45.  The  suspension  of  the  granules 
in  a  drop  of  water  will  facilitate  the  microscopical  examination. 

3.  Solubility. — Try  the  solubility  of  one  form  of  starch  in  each 
of  the  ordinary  solvents  (see  page  23).  If  uncertain  regarding  the 
solubility  in  any  reagent,  filter  and  test  the  filtrate  with  iodine  solu- 
tion as  given  under  5  below.  The  production  of  a  blue  color  would 
indicate  that  the  starch  had  been  dissolved  by  the  solvent. 

4.  Iodine  Test. — Place  a  few  granules  of  starch  in  one  of  the 
depressions  of  a  porcelain  test-tablet  and  treat  with  a  drop  of  a 
dilute  solution  of  iodine  in  potassium  iodide.  The  granules  are 
colored  blue  due  to  the  formation  of  so-called  iodide  of  starch.  The 
cellulose  of  the  granule  is  not  stained  as  may  be  seen  by  examining 
microscopically. 

5.  Iodine  Test  on  Starch  Paste.1 — Repeat  the  iodine  test  using 
the  starch  paste.     Place  2-3  c.c.  of  starch  paste2  in  a  test-tube,  add 

1  Preparation  of  Starch  Paste. — Grind  2  grams  of  starch  powder  in  a  motar 
with  a  small  amount  of  cold  water.  Bring  200  c.c.  of  water  to  the  boiling-point 
and  add  the  starch  mixture  from  the  mortar  with  continuous  stirring.  Bring 
again  to  the  boiling-point  and  allow  it  to  cool.  This  makes  an  approximate  T 
per  cent  starch  paste  which  is  a  very  satisfactory  strength  for  general  use. 

2  For  this  particular  test  a  starch  paste  of  very  satisfactory  strength  may  be 
made  by  mixing  1  c.c.  of  a  1  per  cent  starch  paste  with  100  c.c.  of  water. 


Fig.  7 


CARBOHYDRATES. 

Fig.  8. 


45 


Pea.  Wheat. 

Starch  Granules  from  Various  Sources.     (Leffman  and  Beam.) 


46  PHYSIOLOGICAL    CHEMISTRY. 

a  drop  of  the  dilute  iodine  solution  and  observe  the  production  of  a 
blue  color.  Heat  the  tube  and  note  the  disappearance  of  the  color. 
It  reappears  on  cooling. 

In  similar  tests  note  the  influence  of  alcohol  and  of  alkali  upon 
the  so-called  iodide  of  starch. 

The  composition  of  the  iodide  of  starch  is  not  definitely  known. 

6.  Fehling's  Test. — On  starch  paste  (see  page  27). 

7.  Hydrolysis  of  Starch. — Place  about  25  c.c.  of  starch  paste 
in  a  small  beaker,  add  10  drops  of  concentrated  HC1,  and  boil.  By- 
means  of  a  small  pipette,  at  the  end  of  each  minute,  remove  a  drop 
of  the  solution  to  the  test-tablet  and  make  the  regular  iodine  test. 
As  the  testing  proceeds  the  blue  color  should  gradually  fade  and 
finally  disappear.  At  this  point,  after  cooling  and  neutralizing  with 
solid  KOH,  Fehling's  test  (see  page  27)  should  give  a  positive  result 
due  to  the  formation  of  a  reducing  sugar  from  the  starch.  Make 
the  phenylhydrazine  test  upon  some  of  the  hydrolyzed  starch.  What 
sugar  has  been  formed? 

8.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid 
solution  to  a  small  amount  of  starch  paste  in  a  test-tube.  The  liquid 
will  become  strongly  opaque  and  ordinarily  a  yellowish-white  pre- 
cipitate is  produced.  Compare  this  result  with  the  result  of  the 
similar  experiment  on  dextrin  (page  49). 

9.  Diffusibility  of  Starch  Paste. — Test  the  diffusibility  of  starch 
paste  through  animal  membrane  or  parchment  paper,  making  a 
dialyzer  like  one  of  the  models  shown  in  Fig.  1,  page  25. 

INULIN,  (C6H10O5)x. 

Inulin  is  a  polysaccharide  which  may  be  obtained  as  a  white, 
odorless,  tasteless  powder  from  the  tubers  of  the  artichoke,  elecam- 
pane or  dahlia.  It  has  also  been  prepared  from  the  roots  of  chicory, 
dandelion  and  burdock.  It  is  very  slightly  soluble  in  cold  water 
and  quite  easily  soluble  in  hot  water.  In  cold  alcohol  of  60  per 
cent  or  over  it  is  practically  insoluble.  Inulin  gives  a  negative  reac- 
tion with  iodine  solution.  The  "  yellow  "  color  reaction  with  iodine 
mentioned  in  many  books  is  doubtless  merely  the  normal  color  of 
the  iodine  solution.  It  is  very  difficult  to  prepare  inulin  which  does 
not  reduce  Fehling's  solution  slightly.  This  reducing  power  may 
be  due  to  an  impurity.  Practically  all  commercial  preparations  of 
inulin  possess  considerable  reducing  power. 

Inulin  is  lsevo-rotatory  and  upon  hydrolysis  by  acids  or  by  the 


CARBOHYDRATES.  47 

enzyme  inulase  it  yields  the  monosaccharide  l^evulose  which  readily 
reduces  Fehling's  solution.  The  ordinary  amylolytic  enzymes  occur- 
ring in  the  animal  body  do  not  digest  inulin. 

Experiments  on  Inulin. 

1.  Solubility. — Try  the  solubility  of  inulin  powder  in  each  of 
the  ordinary  solvents.  If  uncertain  regarding  the  solubility  in  any 
reagent,  filter  and  neutralize  the  nitrate  if  it  is  alkaline  in  reaction. 
Add  a  drop  of  concentrated  hydrochloric  acid  to  the  filtrate  and 
boil  it  for  one  minute.  Render  the  solution  neutral  or  slightly 
alkaline  with  solid  potassium  hydroxide  and  try  Fehling's  test. 
What  is  the  significance  of  a  positive  Fehling's  test  in  this  connection  ? 

2.  Iodine  Test. —  (a)  Place  2-3  c.c.  of  the  inulin  solution  in  a 
test-tube  and  add  a  drop  of  dilute  iodine  solution.  What  do  you 
observe  ? 

(b)  Place  a  small  amount  of  inulin  powder  in  one  of  the  depres- 
sions of  a  test-tablet  and  add  a  drop  of  dilute  iodine  solution.  Is 
the  effect  any  different  from  that  observed  above? 

3.  Molisch's  Reaction. — Repeat  this  test  according  to  directions 
given  under  Dextrose,  2,  page  23. 

4.  Fehling's  Test. — Make  this  test  on  the  inulin  solution  accord- 
ing to  the  instructions  given  under  Dextrose,  page  27.  Is  there  any 
reduction?1 

5.  Hydrolysis  of  Inulin. — Place  5  c.c.  of  inulin  solution  in  a 
test-tube,  add  a  drop  of  concentrated  hydrochloric  acid  and  boil 
it  for  one  minute.  Now  cool  the  solution,  neutralize  it  with  con- 
centrated KOH  and  test  the  reducing  action  of  1  c.c.  of  the  solution 
upon  1  c.c.  of  diluted  (1:4)  Fehling's  solution.    Explain  the  result.2 

GLYCOGEN,  (C6H10O5)x. 

(For  discussion  and  experiments  see  Muscular  Tissue,  Chapter 
XV.) 

1  See  the  discussion  of  the  properties  of  inulin,  page  46. 

2  If  the  inulin  solution  gave  a  positive  Fehling  test  in  the  last  experiment  it 
will  be  necessary  to  check  the  hydrolysis  experiment  as  follows :  To  5  c.c.  of 
the  inulin  solution  in  a  test-tube  add  one  drop  of  concentrated  hydrochloric 
acid,  neutralize  with  concentrated  KOH  solution  and  test  the  reducing  action 
of  i  c.c.  of  the  resulting  solution  upon  I  c.c.  of  diluted  (1:4)  Fehling's  solu- 
tion. This  will  show  the  normal  reducing  power  of  the  inulin  solution.  In 
case  the  inulin  was  hydrolyzed,  the  Fehling's  test  in  the  hydrolysis  experiment 
should  show  a  more  pronounced  reduction  than  that  observed  in  the  check 
experiment. 


48  PHYSIOLOGICAL    CHEMISTRY. 

LICHENIN,  (C6H10O5)x. 

Lichenin  may  be  obtained  from  Cetraria  islandica  (Iceland  moss). 
It  forms  a  difficultly  soluble  jelly  in  cold  water  and  an  opalescent 
solution  in  hot  water.  It  is  optically  inactive  and  gives  no  color 
with  iodine.  Upon  hydrolysis  with  dilute  mineral  acids  lichenin 
yields  dextrins  and  dextrose.  It  is  said  to  be  most  nearly  related 
chemically  to  starch.  Saliva,  pancreatic  juice,  malt  diastase  and  gas- 
tric juice  have  no  noticeable  action  on  lichenin. 

DEXTRIN,  (C6H1005)x. 

The  dextrins  are  the  bodies  formed  midway  in  the  stages  of  the 
hydrolysis  of  starch  by  weak  acids  or  an  enzyme.  They  are  amor- 
phous bodies  which  are  easily  soluble  in  water,  acids  and  alkalis 
but  are  insoluble  in  alcohol  or  ether.  Dextrins  are  dextro-rotatory 
and  are  not  fermentable  by  yeast. 

The  dextrins  may  be  hydrolyzed  by  dilute  acids  to  form  dextrose. 
With  iodine  one  form  of  dextrin  (erythro-dextrin)  gives  a  red 
color.     Their  power  to  reduce  Fehling's  solution  is  questioned. 

Experiments  on  Dextrin. 

i.  Solubility. — Test  the  solubility  of  pulverized  dextrin  in  the 
ordinary  solvents  (see  page  23). 

2.  Iodine  Test. — Place  a  drop  of  dextrin  solution  in  one  of  the 
depressions  of  the  test-tablet  and  add  a  drop  of  a  dilute  solution 
of  iodine  in  potassium  iodide.  A  red  color  results  due  to  the  forma- 
tion of  the  red  iodide  of  dextrin.  If  the  reaction  is  not  sufficiently 
pronounced  make  a  stronger  solution  from  pulverized  dextrin  and 
repeat  the  test.  The  solution  should  be  slightly  acid  to  secure  the 
best  results. 

Make  proper  tests  to  show  that  the  red  iodide  of  dextrin  is 
influenced  by  heat,  alkali  and  alcohol  in  a  similar  manner  to  the 
blue  iodide  of  starch  (see  page  46). 

3.  Fehling's  Test. — See  if  the  dextrin  solution  will  reduce  Feh- 
ling's solution. 

4.  Hydrolysis  of  Dextrin. — Take  25  c.c.  of  dextrin  solution  in 
a  small  beaker,  add  5  drops  of  dilute  hydrochloric  acid,  and  boil. 
By  means  of  a  small  pipette,  at  the  end  of  each  minute,  remove  a 
drop  of  the  solution  to  one  of  the  depressions  of  the  test-tablet  and 


CARBOHYDRATES.  49 

make  the  iodine  test.  The  power  of  the  solution  to  produce  a  color 
with  iodine  should  rapidly  disappear.  When  a  negative  reaction  is 
obtained  cool  the  solution  and  neutralize  it  with  concentrated  potas- 
sium hydroxide.  Try  Fehling's  test  (see  page  27).  This  reaction 
is  now  strongly  positive,  due  to  the  formation  of  a  reducing  sugar. 
Determine  the  nature  of  the  sugar  by  means  of  the  phenylhydrazine 
test  (see  pages  24  and  25). 

5.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid 
solution  to  a  small  amount  of  dextrin  solution  in  a  test-tube.  No 
precipitate  forms.  This  result  differs  from  the  result  of  the  similar 
experiment  upon  starch  (see  Starch,  8,  page  46). 

6.  Diffusibility  of  Dextrin. —  (See  Starch,  9,  page  46.) 

7.  Precipitation  by  Alcohol. — To  about  50  c.c.  of  95  per  cent 
alcohol  in  a  small  beaker  add  about  10  c.c.  of  a  concentrated  dextrin 
solution.  Dextrin  is  thrown  out  of  solution  as  a  gummy  white 
precipitate.  Compare  the  result  with  that  obtained  under  Dextrose, 
5,  page  25. 

CELLULOSE,    (C6H10O5)x. 

This  complex  polysaccharide  forms  a  large  portion  of  the  cell 
wall  of  plants.  It  is  extremely  insoluble  and  its  molecule  is  much 
more  complex  than  the  starch  molecule.  The  best  quality  of  filter 
paper  and  the  ordinary  absorbent  cotton  are  good  types  of  cellulose. 

Experiments  on  Cellulose. 

1.  Solubility. — Test  the  solubility  of  cellulose  in  the  ordinary 
solvents  (see  page  23). 

2.  Iodine  Test. — Add  a  drop  of  dilute  iodine  solution  to  a  few 
shreds  of  cotton  on  a  test-tablet.  Cellulose  differs  from  starch  and 
dextrin  in  giving  no  color  with  iodine. 

3.  Formation  of  Amyloid.1 — Add  10  c.c.  of  dilute  and  5  c.c. 
of  concentrated  H2S04  to  some  absorbent  cotton  in  a  test-tube. 
When  entirely  dissolved  (without  heating)  pour  one-half  of  the 
solution  into  another  test-tube,  cool  it  and  dilute  with  water.  Amy- 
loid forms  as  a  gummy  precipitate  and  gives  a  brown  or  blue  colora- 
tion with  iodine. 

After  allowing  the  second  portion  of  the  acid  solution  of  cotton 
to  stand  about  10  minutes,  dilute  it  with  water  in  a  small  beaker  and 
boil  for  15-30  minutes.     Now  cool,  neutralize  with  solid  KOH  and 

1  This  body  derives  its  name  from  amylum  (starch)  and  is  not  to  be  con- 
founded with  amyloid,  the  glycoprotein    (page   106). 

5 


5o 


PHYSIOLOGICAL    CHEMISTRY. 


test  with  Fehling's  solution.     Dextrose  has  been  formed  from  the 
cellulose  by  the  action  of  the  acid. 

4.  Schweitzer's  Solubility  Test. — Place  a  little  absorbent  cotton 
in  a  test-tube,  add  Schweitzer's  reagent,1  and  stir  the  cellulose  with' 
a  glass  rod.  When  completely  dissolved-  acidify  the  solution  with 
acetic  acid.  An  amorphous  precipitate  of  cellulose  is  produced. 
Schweitzer's  reagent  is  the  only  solvent  for  cellulose. 


REVIEW   OF   CARBOHYDRATES. 

In  order  to  facilitate  the  student's  review  of  the  carbohydrates, 
the  preparation  of  a  chart  similar  to  the  appended  model  is  recom- 


MODEL  CHART  FOR  REVIEW  PURPOSES. 


Carbohydrate. 

"o 

t/2 

0> 

H 

01 

a 
-3 
0 

V 

H 

VI 

"11 
u 
0 
0 

s 

QJ 

H 

*u 

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6 

s 
0 

H 

01 

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rt 

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rt  0 
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Dextrose. 
Lsevulose. 





— 

~ 





— 



— 

— 

— 

Maltose. 



Iso-maltose. 

Lactose. 







Sucrose. 

Starch. 



Inulin. 



Glycogen. 

Dextrin. 

Cellulose. 

mended.  The  signs  +  and  —  may  be  conveniently  used  to  indicate 
positive  and  negative  reaction.  Only  those  carbohydrates  which 
are  of  greatest  importance  from  the  standpoint  of  physiological 
chemistry  have  been  included  in  the  chart. 

1  Schweitzer's  reagent  is  made  by  adding  potassium  hydroxide  to  a  solution 
of  cupric  sulphate  which  contains  some  ammonium  chloride.  A  precipitate  of 
cupric  hydroxide  forms  and  this  is  filtered  off,  washed,  and  3  grams  of  the 
moist  cupric  hydroxide  brought  into  solution  in  a  liter  of  20  per  cent  ammonium 
hydroxide. 


CARBOHYDRATES. 


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52  physiological  chemistry. 

"Unknown"  Solutions  of  Carbohydrates. 

At  this  point  the  student  will  be  given  several  "  unknown  "  solu- 
tions, each  solution  containing  one  or  more  ot  the  carbohydrates 
studied.  He  will  be  required  to  detect,  by  means  of  the  tests  on 
the  preceding  pages,  each  carbohydrate  constituent  of  the  several 
"unknown"  solutions  and  hand  in,  to  the  instructor,  a  written 
report  of  his  findings,  on  slips  furnished  by  the  laboratory. 

The  scheme  given  on  page  51  may  be  of  use  in  this  connection. 


CHAPTER    III. 

SALIVARY    DIGESTION. 

The  saliva  is  secreted  by  three  pairs  of  glands,  the  submaxillary, 
sublingual  and  parotid,  reinforced  by  numerous  small  glands  called 
buccal  glands.  The  saliva  secreted  by  each  pair  of  glands  possesses 
certain  definite  characteristics  peculiar  to  itself.  For  instance,  in 
man,  the  parotid  glands  ordinarily  secrete  a  thin,  watery  fluid,  the 
submaxillary  glands  secrete  a  somewhat  thicker  fluid  containing 
mucin,  while  the  product  of  the  sublingual  glands  has  a  more  muci- 
laginous character.  The  saliva  as  collected  from  the  mouth  is  the 
combined  product  of  all  the  glands  mentioned. 

The  saliva  may  be  induced  to  flow  by  many  forms  of  stimuli,  such 
as  chemical,  mechanical,  electrical,  thermal  and  psychical,  the  nature 
and  amount  of  the  secretion  depending,  to  a  limited  degree,  upon 
the  particular  class  of  stimuli  employed  as  well  as  upon  the  character 
of  the  individual  stimulus.  For  example,  in  experiments  upon  dogs 
it  has  been  found  that  the  mechanical  stimulus  afforded  by  dropping 
several  pebbles  into  the  animal's  mouth  caused  the  flow  of  but  one 
or  two  drops  of  saliva,  whereas  the  mechanical  stimulus  afforded 
by  sand  thrown  into  the  mouth  induced  a  copious  flow  of  a  thin 
watery  fluid.  Again,  when  ice-water  or  snow  was  placed  in  the 
animal's  mouth  no  saliva  was  seen,  while  an  acid  or  anything  pos- 
sessing a  bitter  taste,  which  the  dog  wished  to  reject,  caused  a  free 
flow  of  the  thin  saliva.  On  the  other  hand,  when  articles  of  food 
were  placed  in  the  dog's  mouth  the  animal  secreted  a  thicker  saliva 
having  a  higher  mucin  content — a  fluid  which  would  lubricate  the 
food  and  assist  in  the  passage  of  the  bolus  through  the  oesophagus. 
It  was  further  found  that  by  simply  drawing  the  attention  of  the 
animal  to  any  of  the  substances  named  above,  results  were  obtained 
similar  to  those  secured  when  the  substances  were  actually  placed 
in  the  animal's  mouth.  For  example,  when  a  pretense  was  made 
of  throwing  sand  into  the  dog's  mouth,  a  watery  saliva  was  secreted, 
whereas  food  under  the  same  conditions  excited  a  thicker  and  more 
slimy  secretion.  The  exhibition  of  dry  food,  in  which  the  dog  had 
no  particular  interest  (dry  bread)  caused  the  secretion  of  a  large 

53 


54  PHYSIOLOGICAL    CHEMISTRY. 

amount  of  watery  saliva,  while  the  presentation  of  moist  fooclr 
which  was  eagerly  desired  by  the  animal,  called  forth  a  much 
smaller  secretion,  slimy  in  character.  These  experiments  show  it 
to  be  rather  difficult  to  differentiate  between  the  influence  of  physio- 
logical and  psychical  stimuli. 

The  amount  of  saliva  secreted  by  an  adult  in  24  hours  has  been 
variously  placed,  as  the  result  of  experiment  and  observation,  be- 
tween 1000  and  1500  c.c,  the  exact  amount  depending,  among  other 
conditions,  upon  the  character  of  the  food. 

The  saliva  ordinarily  has  a  weak,  alkaline  reaction  to  litmus,  but 
becomes  acid,  in  some  instances,  2-3  hours  after  a  meal  or  during 
fasting.  The  alkalinity  is  due  principally  to  di-sodium  hydrogen 
phosphate  (Na2HP04)  and  its  average  alkalinity  may  be  said  to 
be  equivalent  to  0.08-0.1  per  cent  sodium  carbonate.  The  saliva 
is  the  most  dilute  of  all  the  digestive  secretions,  having  an  average 
specific  gravity  of  1.005  and  containing  only  0.5  per  cent  of  solid 
matter.  Among  the  solids- are  found  albumin,  globulin,  mucin,  urea, 
the  enzymes,  salivary  amylase  (ptyalin)  and  maltase,  phosphates 
and  other  inorganic  constituents.  Potassium  thiocyanate,  KSCN,  is 
also  generally  present  in  the  saliva.  It.  has  been  claimed  that  this 
substance  is  present  in  greatest  amount  in  the  saliva  of  habitual 
smokers.  The  significance  of  thiocyanate  in  the  saliva  is  not  known ; 
it  probably  comes  from  the  ingested  thiocyanates  and  from  the 
breaking  down  of  protein  material. 

The  so-called  tartar  formation  on  the  teeth  is  composed  almost 
entirely  of  calcium  phosphate  with  some  calcium  carbonate,  mucin, 
epithelial  cells  and  organic  debris  derived  from  the  food.  The  cal- 
cium salts  are  held  in  solution  as  acid  salts,  and  are  probably  pre- 
cipitated by  the  ammonia  of  the  breath.  The  various  organic  sub- 
stances just  mentioned  are  carried  down  in  the  precipitation  of  the 
.calcium  salts. 

The  principal  enzyme  of  the  saliva  is  known  as  salivary  amylase 
or  ptyalin.  This  is  an  amylolytic  enzyme  (see  p.  3),  so-called 
because  it  possesses  the  property  of  transforming  complex  carbo- 
hydrates such  as  starch  and  dextrin  into  simpler  bodies.  The 
action  of  salivary  amylase  is  one  of  hydrolysis  and  through  this 
action  a  series  of  simpler  bodies  are  formed  from  the  complex 
starch.  The  first  product  of  the  action  of  the  ptyalin  of  the  saliva 
upon  starch  paste  is  soluble  starch  (amidulin)  and  its  forma- 
tion is  indicated  by  the  disappearance  of  the  opalescence  of  the 
starch    solution.     This   body    resembles   true   starch   in   giving   a 


SALIVARY    DIGESTION.  55 

blue  color  with  iodine.  Next  follows  the  formation,  in  succession, 
of  a  series  of  dextrins,  called  erythro-dextrin,  a-achroo-dcxlrin , 
(3-achroo-dextrin  and.  y-achroo-dextrin,  the  erythro-dextrin  being 
formed  directly  from  soluble  starch  and  later  being  itself  trans- 
formed into  a-achroo-dextriu  from  which  in  turn  are  produced  /?- 
achroo-dextrin  and  y-achroo-dextrin.  Accompanying  each  dextrin 
a  small  amount  of  iso-maltose  is  formed,  the  quantity  of  iso-mal- 
tose  growing  gradually  larger  as  the  process  of  transformation  pro- 
gresses. (Erythro-dextrin  gives  a  red  color  with  iodine,  the  other 
dextrins  give  no  color.)  The  next  stage  is  the  transformation  of 
the  y-achroo-dextrin  into  iso-maltose  and  subsequently  the  transfor- 
mation of  the  iso-maltose  into  maltose,  the  latter  being  the  princi- 
pal end-product  of  the  salivary  digestion  of  starch.  At  this  point 
a  small  amount  of  dextrose  is  formed  from  the  maltose,  through 
the  action  of  the  enzyme  maltase. 

Salivary  amylase  acts  in  alkaline,  neutral  or  combined  acid  solu- 
tions. It  will  act  in  the  presence  of  relatively  strong  combined 
HC1  (see  page  119),  whereas  a  trace  (0.003  Per  cent  to  0.006  per 
cent)  of  ordinary  free  hydrochloric  acid  will  not  only  prevent  the 
action  but  will  destroy  the  enzyme.  By  sufficiently  increasing  the 
alkalinity  of  the  saliva  to  litmus,  the  action  of  the  salivary  amylase 
is  inhibited.  It  has  recently  been  shown  by  Cannon,  that  salivary 
digestion  may  proceed  for  a  considerable  period  after  the  food 
reaches  the  stomach,  owing  to  the  slowness  with  which  the  con- 
tents are  thoroughly  mixed  with  the  acid  gastric  juice  and  the  conse- 
quent tardy  destruction  of  the  enzyme.  Food  in  the  pyloric  end 
of  the  stomach  is  soon  mixed  with  the  gastric  secretion  but  food  in 
the  cardiac  end  is  not  mixed  with  the  acid  gastric  juice  for  a  con- 
siderable period  of  time  and  in  this  region  during  that  time  sali- 
vary digestion  may  proceed  undisturbed. 

Maltase,  sometimes  called  glucase,  is  the  second  enzyme  of  the 
saliva.  It  is  an  amylolytic  enzyme  whose  principal  function  is  the 
splitting  of  maltose  into  dextrose.  Besides  occurring  in  the  saliva 
it  is  also  present  in  the  pancreatic  and  intestinal  juices.  For  experi- 
mental purposes  the  enzyme  is  ordinarily  prepared  from  corn. 
The  principles  of  the  "  reversibility  "  of  enzyme  action  were  first 
demonstrated  in  connection  with  maltase  by  Croft   Hill. 

Microscopical  examination  of  the  saliva  reveals  salivary  corpus- 
cles, bacteria,  food  debris,  epithelial  cells,  mucus  and  fungi.  In 
certain  pathological  conditions  of  the  mouth,  pus  cells  and  blood  cor- 
puscles may  be  found  in  the  saliva. 


56 


PHYSIOLOGICAL    CHEMISTRY. 


Experiments  on  Saliva. 

A  satisfactory  method  of  obtaining  the  saliva  necessary  for  the 
experiments  which  follow  is  to  chew  a  small  piece  of  pure  paraffin 
wax,  thus  stimulating  the  flow  of  the  secretion,  which  may  be  col- 
lected in  a  small  beaker.  Filtered  saliva  is  to  be  used  in  every  ex- 
periment except  for  the  microscopical  examination. 

i.  Microscopical  Examination. — Examine  a  drop  of  unfiltered 
saliva  microscopically  and  compare  with  Fig.   18  below. 

2.  Reaction. — Test  the  reaction  to  litmus. 


Fig.   18. 


^  :%'• 


Microscopical  Constituents  of  Saliva. 

a,  Epithelial  cells ;   b,  salivary  corpuscles ;   c,   fat  drops ;   d,  leucocytes ;   e,  f  and  gx 
bacteria ;  h,  i  and  k,  fission-fungi. 

3.  Specific  Gravity. — Partially  fill  a  urinometer  cylinder  with 
saliva,  introduce  the  urinometer,  and  observe  the  reading. 

4.  Test  for  Mucin. — To  a  small  amount  of  saliva  in  a  test-tube 
add  1-2  drops  of  dilute  acetic  acid.     Mucin  is  precipitated. 

5.  Biuret  Test.1 — Render  a  little  saliva  alkaline  with  an  equal 
volume  of  KOH  and  add  a  few  drops  of  a  very  dilute  (2-5  drops  in 
a  test-tube  of.  water)  cupric  sulphate  solution.  The  formation  of 
a  purplish-violet  color  is  due  to  mucin.  . 

6.  Millon's  Reaction.2 — Add  a  few  drops  of  Millon's  reagent  to 
a  little  saliva.  A  light  yellow  precipitate  formed  by  the  mucin 
gradually  turns  red  upon  being  gently  heated. 

7.  Preparation  of  Mucin. — Pour  25  c.c.  of  saliva  into  100  c.c. 
of  95  per  cent  alcohol,  stirring  constantly.  Cover  the  vessel  and 
allow  the  precipitate  to  stand  at  least  12  hours.  Pour  off  the 
supernatant  liquid,  collect  the  precipitate  on  a  filter  and  wash  it,  in 


The  significance  of  this  reaction  is  pointed  out  on  page  92. 
The  significance  of  this  reaction  is  pointed  out  on  page  90. 


SALIVARY    DIGESTION.  5/ 

turn,  with  alcohol  and  ether.  Finally  dry  the  precipitate,  remove  it 
from  the  paper  and  make  the  following  tests  on  the  mucin:  (a) 
Test  its  solubility  in  the  ordinary  solvents  (see  page  23),  (b) 
Millon's  reaction,  (c)  dissolve  a  small  amount  in  KOH,  and  try 
the  biuret  test  on  the  solution,  (d)  boil  the  remainder,  with  10-25 
c.c.  of  water  to  which  5  c.c.  of  dilute  HC1  has  been  added,  until 
the  solution  becomes  brownish.  Cool,  render  alkaline  with  solid 
KOH,  and  test  by  Fehling's  solution.  A  reduction  should  take 
place.  Mucin  is  what  is  known  as  a  conjugated  protein  or  glyco- 
protein (see  p.  87)  and  upon  boiling  with  the  acid  the  carbohydrate 
group  in  the  molecule  has  been  split  off  from  the  protein  portion 
and  its  presence  is  indicated  by  the  reduction  of  Fehling's  solution. 

8.  Inorganic  Matter. — Test  for  chlorides,  phosphates,  sulphates 
and  calcium.  For  chlorides,  acidify  with  HN03  and  add  AgN03. 
For  phosphates,  acidify  with  HN03,  heat  and  add  molybdic  solu- 
tion.1 For  sulphates,  acidify  with  HC1  and  add  BaCl2  and  warm. 
For  calcium,  acidify  with  acetic  acid,  CH3COOH,  and  add  ammon- 
ium oxalate,  (NH4)2C204. 

9.  Viscosity  Test. — Place  filter  papers  in  two  funnels,  and  to 
each  add  an  equal  quantity  of  starch  paste  (5  c.c).  Add  a  few 
drops  of  saliva  to  one  lot  of  paste  and  an  equivalent  amount  of 
water  to  the  other.  Note  the  progress  of  filtration  in  each  case. 
Why  does  one  solution  filter  more  rapidly  than  the  other? 

10.  Test  for  Nitrites. — Add  1-2  drops  of  dilute  H2S04  to  a 
little  saliva  and  thoroughly  stir.  Now  add  a  few  drops  of  a  potas- 
sium iodide  solution  and  some  starch  paste.  Nitrous  acid  is  formed 
which  liberates  iodine,  causing  the  formation  of  the  blue  iodide  of 
starch. 

11.  Thiocyanate  Tests. —  (a)  Ferric  Chloride  Test, — To  a  little 
saliva  in  a  small  porcelain  crucible,  or  dish,  add  a  few  drops  of 
dilute  ferric  chloride  and  acidify  slightly  with  HC1.  Red  ferric 
thiocyanate  forms.  To  show  that  the  red  coloration  is  not  due  to 
iron  phosphate  add  a  drop  of  HgCl2  when  colorless  mercuric  thio- 
cyanate forms. 

(b)  Solera's  Reaction. — This  test  depends  upon  the  liberation 
of  iodine  through  the  action  of  thiocyanate  upon  iodic  acid.    Moisten 

1  Molybdic  solution  is  prepared  as  follows,  the  parts  being  by  weight : 

1   part,  molybdic  acid. 

4  parts,  ammonium  hydroxide   (Sp.  gr.  0.96). 
15  parts,   nitric   acid    (Sp.   gr.    1.2). 


58  PHYSIOLOGICAL    CHEMISTRY. 

a  strip  of  starch  paste-iodic  acid  test  paper1  with  a  little  saliva.  If 
thiocyanate  be  present  the  test  paper  will  assume  a  blue  color,  due 
to  the  liberation  of  iodine  and  the  subsequent  formation  of  the 
so-called  iodide  of  starch. 

12.  Digestion  of  Starch  Paste. — To  25  c.c.  of  starch  paste  in 
a  small  beaker,  add  5  drops  of  saliva  and  stir  thoroughly.  At  in- 
tervals of  a  minute  remove  a  drop  of  the  solution  to  one  of  the  de- 
pressions in  a  test-tablet  and  test  by  the  iodine  test.  If  the  blue 
color  with  iodine  still  forms  after  5  minutes,  add  another  5  drops 
of  saliva.  The  opalescence  of  the  starch  solution  should  soon  dis- 
appear, indicating  the  formation  of  soluble  starch  which  gives  a  blue 
color  with  iodine.  This  body  should  soon  be  transformed  into 
erythro-dextrin  which  gives  a  red  color  with  iodine  and  this  in  turn 
should  pass  into  achroo-dextrin  which  gives  no  color  with  iodine. 
This  is  called  the  achromic  point.  When  this  point  is  reached  test 
by  Fehling's  test  to  show  the  production  of  a  reducing  body.  A 
positive  Fehling's  test  may  be  obtained  while  the  solution  still 
reacts  red  with  iodine  inasmuch  as  some  iso-maltose  is  formed 
from  the  soluble  starch  coincidently  with  the  formation  of  the 
erythro-dextrin.  How  long  did  it  take  for  a  complete  transforma- 
tion of  the  starch? 

13.  Digestion  of  Dry  Starch. — In  a  test-tube  shake  up  a  small 
amount  of  dry  starch  with  a  little  water.  Add  a  few  drops  of 
saliva,  mix  well  and  allow  to  stand.  After  10-20  minutes  filter  and 
test  the  filtrate  by  Fehling's  test.     What  is  the  result  and  why  ? 

14.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a  test- 
tube  add  10  drops  of  saliva  and  place  the  tube  in  the  water-bath  at 
400  C.  After  one-half  hour  test  the  solution  by  Fehling's  test.2 
Is  any  reducing  substance  present  ?  What  do  you  conclude  regard- 
ing the  salivary  digestion  of  inulin? 

15.  Influence  of  Temperature. — In  each  of  four  tubes  place 
about  5  c.c.  of  starch  paste.  Immerse  one  tube  in  cold  water  from 
the  faucet,  keep  a  second  at  room  temperature  and  place  a  third 
on  the  water-bath  at  40 °  C.  Now  add  to  the  contents  of  each 
of  these  three  tubes  two  drops  of  saliva  and  shake  well;  to  the 

1  This  test  paper  is  prepared  as  follows :  Saturate  a  good  quality  of  filter  paper 
with  0.5  per  cent  starch  paste  to  which  has  been  added  sufficient  iodic  acid  to 
make  a  1  per  cent  solution  of  iodic  acid  and  allow  the  paper  to  dry  in  the  air. 
Cut  it  in  strips  of  suitable  size  and  preserve  for  use. 

2  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the  saliva 
it  will  be  necessary  to  determine  the  extent  of  the  original  reduction  by  means 
of  a  "check"  test  (see  page  47). 


SALIVARY    DIGESTION.  59 

contents  of  the  fourth  tube  add  two  drops  of  boiled  saliva.  Test 
frequently  by  the  iodine  test,  using  the  test-tablet,  and  note  in  which 
tube  the  most  rapid  digestion  occurs.     Explain  the  results. 

16.  Influence  of  Dilution. — Take  a  series  of  6  test-tubes  each 
containing  9  c.c.  of  water.  Add  1  c.c.  of  saliva  to  tube  1  and  shake 
thoroughly.  Remove  1  c.c.  of  the  solution  from  tube  1  to  tube 
2  and  after  mixing'  thoroughly  remove  1  c.c.  from  tube  2  to  tube  3. 
Continue  in  this  manner  until  you  have  6  saliva  solutions  of  gradu- 
ally decreasing  strength.  Now  add  starch  paste  in  equal  amounts 
to  each  tube,  mix  very  thoroughly  and  place  on  the  water-bath  at 
400  C.  After  10-20  minutes  test  by  both  the  iodine  and  Feh- 
ling's tests.     In  how  great  dilution  does  your  saliva  act? 

17.  Influence  of  Acids  and  Alkalis. —  (a)  Influence  of  Free 
Acid. — Prepare  a  series  of  six  tubes  in  each  of  which  is  placed  4 
c.c.  of  one  of  the  following  strengths  of  free  HC1 :  0.2  per  cent, 
o.  1  per  cent,  0.05  per  cent,  0.025  per  cent,  0.0125  per  cent  and 
0.006  per  cent.  Now  add  2  c.c.  of  starch  paste  to  each  tube  and 
shake  them  thoroughly.  Complete  the  solutions  by  adding  2  c.c. 
of  saliva  to  each  and  repeat  the  shaking.  The  total  acidity  of  this 
series  would  be  as  follows:  0.1  per  cent,  0.05  per  cent,  0.025  per 
cent,  0.0125  per  cent,  0.006  per  cent  and  0.003  Per  cent-  Place 
these  tubes  on  the  water-bath  at  40°  C.  for  10-20  minutes.  Divide 
the  contents  of  each  tube  into  two  parts,  testing  one  part  by  the 
iodine  test  and  testing  the  other,  after  neutralization,  by  Fehling's 
test.     What  do  you  find? 

(b)  Influence  of  Combined  Acid. — Repeat  the  first  three  experi- 
ments of  the  above  series  using  combined  hydrochloric  acid  (see 
page  119)  instead  of  the  free  acid.  How  does  the  action  of  the 
combined  acid  differ  from  that  of  the  free  acid? 

(c)  Influence  of  Alkali. — Repeat  the  first  four  experiments  under 
(a)  replacing  the  HC1  by  2  per  cent,  1  per  cent,  0.5  per  cent  and 
0.25  per  cent  Na2C03.  Neutralize  the  alkalinity  before  trying  the 
iodine  test  (see  Starch,  5,  page  44). 

(d)  Nature  of  the  Action  of  Acid  and  Alkali. — Place  2  c.c.  of 
saliva  and  2  c.c.  of  0.2  per  cent  HC1  in  a  test-tube  and  leave  for 
15  minutes.  Neutralize  the  solution,  add  4  c.c.  of  starch  paste 
and  place  the  tube  on  the  water-bath  at  400  C.  In  10  minutes 
test  by  the  iodine  and  Fehling's  tests  and  explain  the  result.  Repeat 
the  experiment,  replacing  the  0.2  per  cent  HC1  by  2  per  cent 
Na2C03.     What  do  you  deduce  from  these  two  experiments? 

18.  Influence  of  Metallic  Salts,  etc. — In  each  of  a  series  of 


60  PHYSIOLOGICAL    CHEMISTRY. 

tubes  place  4  c.c.  of  starch  paste  and  x/z  c.c.  of  one  of  the  solutions 
named  below.  Shake  well,  add  ^  c.c.  of  saliva  to  each  tube,  thor- 
oughly mix,  and  place  on  the  water-bath  at  400  C.  for  10-20 
minutes.  Show  the  progress  of  digestion  by  means  of  the  iodine 
and  Fehling  tests.  Use  the  following  chemicals :  Metallic  salts, 
10  per  cent  plumbic  acetate,  2  per  cent  cupric  sulphate,  5  per  cent 
ferric  chloride,  8  per  cent  mercuric  chloride;  Neutral  salts,  10  per 
cent  sodium  chloride,  10  per  cent  magnesium  sulphate,  3  per  cent 
barium  chloride,  10  per  cent  Rochelle  salt.  Also  try  the  influence 
of  2  per  cent  carbolic  acid,  95  per  cent  alcohol,  and  ether  and  chlor- 
oform.    What  are  your  conclusions? 

19.  Excretion  of  Potassium  Iodide. — Ingest  a  small  dose  of 
potassium  iodide  (0.2  gram)  contained  in  a  gelatin  capsule,  quickly 
rinse  out  the  mouth  with  water  and  then  test  the  saliva  at  once  for 
iodine.  This  test  should  be  negative.  Make  additional  tests  for 
iodine  at  2  minute  intervals.  The  test  for  iodine  is  made  as  fol- 
lows :  Take  1  c.c.  of  NaN02  and  1  c.c.  of  dilute  HoSCV  in  a  test- 
tube,  add  a  little  saliva  directly  from  the  mouth,  and  a  small  amount 
of  starch  paste.  If  convenient,  the  urine  may  also  be  tested.  The 
formation  of  a  blue  color  signifies  that  the  potassium  iodide  is 
being  excreted  through  the  salivary  glands.  Note  the  length  of 
time  elapsing  between  the  ingestion  of  the  potassium  iodide  and 
the  appearance  of  the  first  traces  of  the  substance  in  the  saliva. 
The  chemical  reactions  taking  place  in  this  experiment  are  indicated 
in  the  following  equations : 

( a)  2NaN02  +  H2S04  =  2HN02<  +  Na2S04. 

( b )  2KI  +  H2S04  =  2HI  +  K2S04. 

(c)  2HN02  +  2HI  =  I2  +  2H20  +  2NO. 

20.  Qualitative  Analysis  of  the  Products  of  Salivary  Diges- 
tion.— To  25  c.c.  of  the  products  of  salivary  digestion  (saved  from 
Experiment  12  or  furnished  by  the  instructor),  add  100  c.c.  of 
95  per  cent  alcohol.  Allow  to  stand  until  the  white  precipitate  has 
settled.  Filter,  evaporate  the  filtrate  to  dryness,  dissolve  the  resi- 
due in  5-10  c.c.  of  water  and  try  Fehling's  test  (page  27)  and  the 
phenylhydrazine  reaction  (see  Dextrose,  3,  page  24).  On  the  dex- 
trin precipitate  try  the  iodine  test  (page  44).  Also  hydrolyze  the 
dextrin  as  given  under  Dextrin,  4,  page  48. 

1  Instead  of  this  mixture  a  few  drops  of  HNOs  possessing  a  yellowish  or 
brownish  color  due  to  the  presence  of  HNO2  may  be  employed. 


CHAPTER  IV. 

PROTEINS.1      THEIR   DECOMPOSITION   AND 
SYNTHESIS. 

The  proteins  are  a  class  of  substances,  which  in  the  light  of  our 
present  knowledge,  consist,  in  the  main  of  combinations  of  a- 
amino-acids  or  their  derivatives.  These  protein  substances  form 
the  chief  constituents  of  many  of  the  fluids  of  the  body,  constitute 
the  organic  basis  of  animal  tissue,  and  at  the  same  time  occupy  a 
decidedly  preeminent  position  among  our  organic  food-stuffs.  They 
are  absolutely  necessary  to  the  uses  of  the  animal  organism  for  the 
continuance  of  life  and  they  cannot  be  satisfactorily  replaced  in  the 
diet  of  such  an  organism  by  any  other  dietary  constituent  either 
organic  or  inorganic.  Such  an  organism  may  exist  without  pro- 
tein food  for  a  period  of  time,  the  length  of  the  period  varying 
according  to  the  specific  organism  and  the  nature  of  the  substitu- 
tion offered  for  the  protein  portion  of  the  diet.  Such  a  period  is, 
however,  distinctly  one  of  existence  rather  than  one  of  normal  life 
and  one  which  is  consequently  not  accompanied  by  such  a  full  and 
free  exercise  of  the  various  functions  of  the  organism  as  would  be 
possible  upon  an  evenly  balanced  ration,  i.  c.,  one  containing  the 
requisite  amount  of  protein  food.  These  protein  substances,  are. 
furthermore,  essential  constituents  of  all  living  cells  and  therefore 
without  them  vegetable  life  as  well  as  animal  life  is  impossible. 

The  proteins,  which  constitute  such  an  important  group  of  sub- 
stances, differ  from  the  carbohydrates  and  fats  very  decidedly  in 
elementary  composition.  In  addition  to  containing  carbon,  hy- 
drogen, and  oxygen,  which  are  present  in  fats  and  carbohydrates, 
the  proteins  invariably  contain  nitrogen  in  their  molecule  and  gen- 
erally sulphur  also.  Proteins  have  also  been  identified  which  con- 
tain phosphorus,  iron,  copper,  iodine,  manganese,  and  sine.  The 
percentage  composition  of  the  more  important  members  of  the 
group  of  protein  substances  would  fall  within  the  following  limits : 
C  =  50-55  per  cent,  H=  6-7.3  Per  cent-  0  =  19-24  per  cent, 
N=  15-19  per  cent.  5  =  0.3-2.5  per  cent,  P  =  0.4-0.8  per  cent 

1  The  term  proteid  has  been  very  widely  used  by  English-speaking  scientists 
to  signify  the  class  of  substances  we  have  called  proteins. 

61 


62  PHYSIOLOGICAL    CHEMISTRY. 

when  present.  When  iron,  copper,  iodine,  manganese,  or  zinc  are 
present  in  the  protein  molecule  they  are  practically  without  excep- 
tion present  only  in   traces} 

Of  all  the  various  elements  of  the  protein  molecule,  nitrogen  is 
by  far  the  most  important.  The  human  body  needs  nitrogen  for 
the  continuation  of  life,  but  it  cannot  use  the  nitrogen  of  the  air 
or  that  in  various  other  combinations  as  we  find  it  in  nitrates, 
nitrites,  etc.  However,  in  the  protein  molecule  the  nitrogen  is  pres- 
ent in  a  form  which  is  utilizable  by  the  body.  The  nitrogen  in 
the  protein  molecule  occurs  in  at  least  four  different  forms  as 
follows : 

I.  Monamino  acid  nitrogen. 
II.  Diamino  acid  nitrogen  or  basic  nitrogen. 

III.  Amide  nitrogen. 

IV.  A  guanidine  residue. 

The  actual  structure  of  the  protein  molecule  is  still  unknown, 
and  we  have  as  yet  no  means  by  which  its  molecular  weight  can 
be  even  approximately  established.  The  many  attempts  which 
have  been  made  to  determine  this  have  led  to  very  different  results, 
some  of  which  are  given  in  the  following  table: 

Serum  albumin   =  4572-5 100-5 135 
Egg  albumin        =  4900-6542 
Globin  =  1 5000-16086 

Oxyhemoglobin  =  1 4800-1 5000-1 665  5-1 6730 

Of  these  figures,  those  given  for  oxyhemoglobin  deserve  the 
most  consideration,  for  these  are  based  on  the  atomic  ratios  of  the 
sulphur  and  iron  contained  in  this  substance.  The  simplest  formula 
that  can  be  calculated  from  analyses  of  oxyhemoglobin,  namely, 
C658H1181N207S2FeO210,  serves  to  show  the  great  complexity  of  this 
substance.  The  following  formulas  which  have  been  proposed  for 
typical  protein  substances  may  serve  to  further  impress  the  fact  of 
the  great  size  of  the  protein  molecule  : 

Egg  albumin      =  C239H386N5SS207S 
Serum  albumin  =  C450H720N11GS6O140 

The  decomposition2  of  protein  substances  may  be  brought  about 

1  Some  investigators  regard  these  elements  as  contaminations,  or  constituents 
of   some  non-protein  substance   combined  with  the  protein. 

2  The  terms  "  degradation,"  "  dissociation,"  and  "  cleavage,"  are  often  used 
in  this  connection. 


PROTEINS. 


63 


by  oxidation  or  hydrolysis,  but  inasmuch  as  the  hydrolytic  proce- 
dure has  been  productive  of  the  more  satisfactory  results,  that  type 
of  decomposition  procedure  alone  is  used  at  present.  This  hydrolysis 
of  the  protein  molecule  may  be  accomplished  by  acids,  alkalis,  or 
superheated  steam,  and  in  digestion  by  the  action  of  the  proteolytic 
enzymes.  The  character  of  the  decomposition  products  varies  ac- 
cording to  the  method  utilized  in  tearing  the  molecule  apart.  Bear- 
ing this  in  mind,  we  may  say  that  the  decomposition  products  of 
proteins  include  proteoses,  peptones,  peptides,  carbon  dioxide,  am- 
monia, hydrogen  sulphide,  and  amino  acids.  These  amino  acids 
constitute  a  long  list  of  important  substances  which  contain  nuclei 
belonging  either  to  the  aliphatic,  carbocyclic,  or  heterocyclic  series. 
The  list  includes,  glycocoll,  alanine,  serine,  phenylalanine,  tyrosine, 
cystine,  tryptophane,  histidine,  valine,  arginine,  leucine,  isoleucine, 
lysine,  aspartic  acid,  glutamic  acid,  proline,  oxy proline,  and  diamino- 
trihydroxydodecanoic  acid.  Of  these  amino  acids,  tyrosine  and 
phenylalanine  contain  carbocyclic  nuclei,  histidine,  proline  and  tryp- 
tophane contain  heterocyclic  nuclei,  and  the  remaining  members  of 
the  list,  as  given,  contain  aliphatic  nuclei.  The  amino  acids  are  pre- 
eminently the  most  important  class  of  protein  decomposition  prod- 
ucts. These  amino  acids  are  all  a-amino  acids,  and,  with  the 
exception  of  glycocoll,  are  all  optically  active.  Furthermore  they 
are  amphoteric  substances  and  consequently  are  able  to  form  salts 
with  both  bases  and  acids.  These  properties  are  inherent  in  the 
NH2  and  COOH  groups  of  the  amino  acids. 

The  decomposition  products  of  protein  may  be  grouped  as  pri- 
mary and  secondary  decomposition  products.  By  primary  products 
are  meant  those  which  exist  as  radicals  within  the  protein  molecule 
and  which  are  liberated,  upon  cleavage  of  this  molecule,  with  their 
carbon  chains  intact  and  the  position  of  their  nitrogen  unaltered. 
The  secondary  products  are  those  which  result  from  the  disintegra- 
tion of  the  primary  cleavage  products.  No  matter  what  method 
is  used  to  decompose  a  given  protein  molecule,  the  primary  products 
are  largely  the  same  under  all  conditions.1 

In  the  process  of  hydrolysis  the  protein  molecule  is  gradually 
broken  dow.n  and  less  complicated  aggregates  than  the  original 
molecule  are  formed,  which  are  known  as  proteoses,  peptones  and 
peptides  and  which  still  possess  true  protein  characteristics.  Fur- 
ther hydrolysis  causes  the  ultimate  transformation  of  these  sub- 

1  Alkaline  hydrolysis  yields  urea  and  ornithine  which  result  from  arginine, 
the  product  of  acid  hydrolysis. 


64  PHYSIOLOGICAL    CHEMISTRY. 

stances,  of  a  protein  nature,  into  the  amino  acids  of  known  chemi- 
cal structure.  In  this  decomposition  the  protein  molecule  is  not 
broken  down  in  a  regular  manner  into  y2,  Ya,  %  portions  and 
the  amino  acids  formed  in  a  group  at  the  termination  of  the  hy- 
drolysis. On  the  contrary,  certain  amino  acids  are  formed  very 
early  in  the  process,  in  fact  while  the  main  hydrolytic  action  has 
proceeded  no  further  than  the  proteose  stage.  Gradually  the  com- 
plexity of  the  protein  portion  undergoing  decomposition  is  sim- 
plified by  the  splitting  off  of  the  amino  acids  and  finally  it  is  so  far 
decomposed  through  previous  cleavages  that  it  yields  only  amino 
acids  at  the  succeeding  cleavage.  In  short  the  general  plan  of  the 
hydrolysis  of  the  protein  molecule  is  similar  to  the  hydrolysis  of 
starch.  In  the  case  of  starch  there  is  formed  a  series  of  dextrins  of 
gradually  decreasing  complexity  and  coincidently  with  the  formation 
of  each  dextrin  a  small  amount  of  sugar  is  split  off  and  finally 
nothing  but  sugar  remains.  In  the  case  of  protein  hydrolysis  there 
is  a  series  of  proteins  of  gradually  decreasing  complexity  produced 
and  coincidently  with  the  formation  of  each  new  protein  substance 
amino  acids  are  split  off  and  finally  the  sole  products  remaining 
are  amino  acids. 

Inasmuch  as  diversity  in  the  method  of  decomposing  a  given 
protein  does  not  result  in  an  equally  diversified  line  of  decomposition 
products,  but,  on  the  other  hand,  yields  products  which  are  quite 
comparable  in  character,  it  may  be  argued  that  there  are  probably 
well  denned  lines  of  cleavage  in  the  individual  protein  molecule 
and  that  no  matter  what  the  force  brought  to  bear  to  tear  such  a 
molecule  apart,  the  disintegration,  when  it  comes,  will  yield  in 
every  case,  certain  definite  fragments.  These  fragments  may  be 
called  the  "  building  stones  "  of  the  protein  molecule,  a  term  used 
by  some  of  the  German  investigators.  Take,  for  example,  the 
decomposition  of  protein  which  may  be  brought  about  through 
the  action  of  the  enzyme  trypsin  of  the  pancreatic  juice.  When 
this  enzyme  is  allowed  to  act  upon  a  given  protein,  the  latter  is 
disintegrated  in  a  series  of  definite  cleavages,  resulting  in  the  for- 
mation of  proteoses,  peptones  and  peptides  in  regular  order,  the 
peptides  being  the  last  of  the  decomposition  products  which  possess 
protein  characteristics.  They  are  all  built  up  from  amino  acids  and 
are  therefore  closely  related  to  these  acids  on  the  one  side  and  to 
peptones  on  the  other.  We  have  di-,  tri-,  tetra-,  penta-,  deca-,  and 
poly-peptides  which  are  named  according  to  the  number  of  amino 
acids   included   in   the  peptide   molecule.     Following  the  peptides 


PROTEINS.  65 

there  are  a  diverse  assortment  of  monamino  and  diamino  acids 
which  constitute  the  final  products  of  the  protein  decomposition. 
These  acids  are  devoid  of  any  protein  characteristics  and  are  there- 
fore decidedly  different  from  the  original  substance  from  which 
they  were  derived.  From  a  protein  of  huge  molecular  weight,  a 
typical  colloid,  perhaps  but  slightly  soluble,  and  entirely  non-dif- 
fusible, we  have  passed  by  way  of  proteoses,  peptones,  and  pep- 
tides to  a  class  of  simpler  crystalline  substances  which  are,  for  the 
most  part,  readily  soluble  and  diffusible. 

These  amino  acids  after  their  production  in  the  process  of  diges- 
tion, as  just  indicated,  are  synthesized  within  the  organism  to  form 
protein  material  which  goes  to  build  up  the  tissues  of  the  body. 
It  is  thus  seen  that  the  amino  acids  are  of  prime  importance  in  the 
animal  economy.  Moreover,  it  is  important  to  remember  that  these 
essential  factors  in  metabolism  and  nutrition  cannot  be  produced 
within  the  animal  organism  from  their  elements,  but  are  only  yielded 
upon  the  hydrolysis  of  ingested  protein  of  animal  or  vegetable  origin. 

When  we  examine  the  formulas  of  the  principal  members  of  the 
crystalline  end-products  of  protein  decomposition  we  note  that  they 
are  invariably  acids,  as  has  already  been  mentioned,  and  contain  an 
NH2  group  in  the  a  position.  This  relation  of  the  NH2  group  to 
the  acid  radical  is  constant,  no  matter  what  other  groups  or  radicals 
are  present.  We  may  have  straight  chains  as  in  alanine  and  glu- 
tamic acid,  the  benzene  ring  as  in  phenylalanine  or  we  may  have 
sulphurised  bodies  as  in  cystine  and  still  the  formula  is  always  of 
the  same  type,  i.  e., 

NH2 

I 
E  -  CH  -  COOH 

It  is  seen  that  this  characteristic  grouping  in  the  amino  acid  pro- 
vides each  one  of  these  ultimate  fragments  of  the  protein  molecule 
with  both  a  strong  acid  and  a  strong  basic  group.  For  this  reason 
it  is  theoretically  possible  for  a  large  number  of  these  amino  acids 
to  combine  and  the  resulting  combinations  may  be  very  great  in 
number,  since  there  is  such  a  varied  assortment  of  the  acids.  The 
protein  molecule,  which  is  of  such  mammoth  proportions,  is  prob- 
ably constructed  on  a  foundation  of  this  sort.  Of  late  much  valu- 
able data  have  been  collected  regarding  the  synthetic  production  of 
protein  substances,  the  leaders  in  this  line  of  investigation  being 
Fischer  and  Abderhalden.  After  having  gathered  a  mass  of  data 
6 


66  PHYSIOLOGICAL    CHEMISTRY. 

regarding  the  final  products  of  the  protein  decomposition  and 
demonstrating  that  amino  acids  were  the  ultimate  results  of  the 
various  forms  of  decomposition,  these  investigators,  and  notably 
Fischer,  set  about  in  an  effort  to  form,  from  these  amino  acids,  by- 
synthetic  means,  substances  which  should  possess  protein  character- 
istics. The  simplest  of  these  bodies  formed  in  this  way  was  synthe- 
sized from  two  molecules  of  glycocoll  with  the  liberation  of  water, 
thus: 


CH9  -  NEL  -  CO  iOH  Hi  HN  -  CEL  -  COOH. 


L2  -L-N  J.O-2 


The  body  thus  formed  is  a  dipeptide,  called  glycyl-glycine.  In  an 
analogous  manner  may  be  produced  leucyl-leucine ,  through  the 
synthesis  of  two  molecules  of  leucine  or  leucyl-alanyl- glycine  through 
the  union  of  one  molecule  of  leucine,  one  of  alanine,  and  one  of 
glycocoll.  By  this  procedure  Fischer  and  his  pupils  have  been  able 
to  make  a  large  number  of  peptides  containing  varied  numbers  of 
amino  acid  radicals,  the  name  polypeptides  being  given  to  the  whole 
group  of  synthetic  substances  thus  formed.  The  most  complex  poly- 
peptide yet  produced  is  one  containing  fifteen  glycocoll  and  three 
leucine  residues. 

Notwithstanding  the  fact  that  most  synthetic  polypeptides  are 
produced  through  a  union  of  amino  acids  by  means  of  their  imide 
bonds,  it  must  not  be  imagined  that  the  protein  molecule  is  con- 
structed from  amino  acids  linked  together  in  straight  chains  in  a 
manner  analogous  to  the  formation  of  simple  peptides,  such  as 
glycyl-glycine.  The  molecular  structure  of  the  proteins  is  much  too 
complex  to  be  explained  upon  any  such  simple  formation  as  that. 
There  must  be  a  variety  of  linkings,  since  there  is  a  varied  assort- 
ment of  decomposition  products  of  totally  different  structure. 

Many  of  these  synthetic  bodies  respond  to  the  biuret  test,  are 
precipitated  by  phosphotungstic  acid  and  behave,  in  other  ways, 
as  to  leave  no  doubt  as  to  their  protein  characteristics.  For  instance, 
a  number  of  amino  acids  each  possessing  a  sweet  taste  have  been 
synthesized  in  such  a  manner  as  to  yield  a  polypeptide  of  bitter 
taste,  a  well  known  characteristic  of  peptones.  From  the  fact  that 
the  polypeptides  formed  in  the  manner  indicated  have  free  acidic 
and  basic  radicals  we  gather  the  explanation  of  the  amphoteric 
character  of  true  proteins.  Fischer  expresses  the  encouraging 
belief  that  he  will  soon  be  able  to  produce  a  true  protein  by  the 
synthesis  of  its  decomposition  products.  Silk  fibroin  is  the  protein 
substance  he  expects  to  synthesize.     He  no  doubt  will  perform  this 


PROTEINS.  67 

joint  office  for  organic  and  physiological  chemistry  if  it  is  capable 
of  performance  by  the  present  methods  of  technique.  Even  Fischer, 
however,  is  frank  enough  to  say  that  the  production  of  the  great 
body  of  protein  substances  synthetically,  will,  under  the  most  en- 
couraging conditions,  be  a  terrific  task,  involving  the  "  life-work  of 
a  whole  army  of  inventive  and  diligent  chemists." 

For  the  benefit  of  those  especially  interested  in  such  matters  a 
photograph  of  the  Fischer  apparatus  (Fig.  22,  page  71)  used  in 
the  fractional  distillation,  in  vacuo,  of  the  esters  of  the  decompo- 
sition products  of  the  proteins,  as  well  as  micro-photographs  and 
drawings  of  preparations  of  several  of  these  decomposition  products 
(Figs  19  to  31,  pages  68  to  81)  are  introduced.  For  the  prepara- 
tions and  the  photograph  of  the  apparatus  the  author  is  indebted 
to  Dr.  T.  B.  Osborne,  of  New  Haven,  Conn.,  who  has  made  many 
important  observations  upon  the  hydrolysis  of  proteins.  The  repro- 
duction of  the  crystalline  form  of  some  of  the  more  recent  of  the 
products  may  be  of  interest  to  those  viewing  the  field  of  physio- 
logical chemistry  from  other  than  the  student's  aspect. 

An  extended  discussion  of  the  various  decomposition  products 
being  out  of  place  in  a  book  of  this  character,  we  will  simply  make 
a  few  general  statements  in  connection  with  the  primary  decompo- 
sition products. 

DISCUSSION   OF  THE   PRODUCTS. 

Ammonia,  NH3. — Ammonia  is  an  important  decomposition 
product  of  all  proteins  and  probably  arises  from  an  amide  group 
combined  with  a  carboxyl  group  of  some  of  the  amino  acids.  It 
is  possible  that  the  dibasic  acids,  aspartic  and  glutamic,  furnish 
most  of  these  carboxyl  groups.  This  is  indicated  by  the  more  or 
less  close  relationship  which  exists  between  the  amount  of  ammonia 
and  that  of  the  dibasic  acids  which  the  several  proteins  yield  upon 
decomposition.  The  elimination  of  the  ammonia  from  proteins 
under  the  action  of  acids  and  alkalis  is  very  similar  to  that  from 
amides  like  asparagine. 

Glycocoll,  C2H5N02. — Glycocoll,  or  amino  acetic  acid,  is  the 
simplest  of  the  amino  acids  and  has  the  following  formula : 

NH2 

H-C-COOH. 

I 
H 


68 


PHYSIOLOGICAL    CHEMISTRY. 


Glycocoll,  as  the  formula  shows,  contains  no  asymmetric  carbon  atom, 
and  is  the  only  amino  acid  yielded  by  protein  decomposition  which 
is  optically  inactive.  Glycocoll  and  leucine  were  the  first  decom- 
position products  of  proteins  to  be  discovered  (1820).  Upon  ad- 
ministering benzoic  acid  to  animals  the  output  of  hippuric  acid 
in  the  urine  is  greatly  increased,  thus  showing  a  synthesis  of  benzoic 
acid  and  glycocoll  in  the  organism  (see  p.  160,  Chapter  IX) .  Glyco- 
coll, ingested  in  small  amount,  is  excreted  in  the  urine  as  urea, 
whereas  if  administered  in  excess  it  appears  in  part  unchanged  in 
the  urine.  It  is  usually  separated  from  the  mixture  of  protein  de- 
composition products  as  the  hydrochloride  of  the  ester.  The  crys- 
talline form  of  this  compound  is  shown  in  Fig.  19. 


Fig.  19. 


Glycocoll  Ester  Hydrochloride. 

Alanine,  C3H7N02. — Alanine  is  a-amino-propionic  acid,  and  as 
such  it  may  be  represented  structurally  as  follows : 

H    NH2 

H-C-C-COOH. 


H    H 

Obtained  from  protein  substances,  alanine  is  dextro-rotatory,  is 
very  soluble  in  water,  and  possesses  a  sweet  taste.  Tyrosine, 
phenylalanine,  cystine  and  serine  are  derivatives  of  alanine.  This 
amino  acid  has  been  obtained  from  nearly  all  proteins  examined. 
Its  absence,  from  those  proteins  from  which  it  has  not  been  obtained. 


PROTEINS. 


69 


has  not  been  proven.     Most  proteins  yield  relatively  small  amounts 
of  alanine. 

Serine,  C3H7N03. — Serine  is  a-amino-P-hydroxy-propionic  acid 
and  possesses  the  following  structural  formula  : 

OH  NH2 

H  _  C  -  C  -  COOH 

I         I 
H      H 

Fig.  20. 


Serine. 

Serine  obtained  from  proteins  is  laevo-rotatory,  possesses  a  sweet 
taste  and  is  quite  soluble  in  water.  Serine  is  not  obtained  in  quantity 
from  most  proteins  but  is  yielded  abundantly  by  silk  glue.  Owing 
to  the  difficulty  of  separating  serine  it  has  not  been  found  in  a 
number  of  proteins  in  which  it  probably  occurs.  Serine  crystals  are 
shown  in  Fig.  20,  above. 

Phenylalanine,  C9HuN02.  — This  product  is  phenyl-a-amino- 
propionic  acid,  and  may  be  represented  graphically  as  follows : 

H    NHo 


-C-C-COOH. 
H   H 


The  laevo-rotatory  form  is  obtained  from  proteins.     Phenylalanine 
has  been  obtained  from  all  the  proteins  examined  except  from  the 


yo 


PHYSIOLOGICAL    CHEMISTRY. 


protamines  and  some  of  the  albuminoids.     The  yield  of  this  body 
from  the  decomposition  of  proteins  is  frequently  greater  than  the 


Fin.  21. 


Phenylalanine. 

yield  of  tyrosine.     The  crystalline  form  of  phenylalanine  is  shown 
in  Fig.  21. 

Tyrosine,  CgH^NC^. — Tyrosine,  one  of  the  first  discovered  end- 
products  of  protein  decomposition,  is  the  amino  acid,  p-oxyphenyl- 
a-amino-propionic  acid.     It  has  the  following  formula : 

H    NH2 

I       I 
_C-C-COOH. 


H   H 


OH 


The  tyrosine  which  results  from  protein  decomposition  is  usually 
lsevo-rotatory  although  the  dextro-rotatory  form  sometimes  occurs. 
Tyrosine  is  one  of  the  end-products  of  tryptic  digestion  and  usually 
separates  in  conspicuous  amount  early  in  the  process  of  digestion. 
It  does  not  occur,  however,  as  an  end-product  of  the  decomposition 
of  gelatin. 

Tyrosine  is  found  in  old  cheese,  and  derives  its  name  from  this  fact. 
It  crystallizes  in  tufts,  sheaves  or  balls  of  fine  needles,  which  decom- 
pose at  295 °  C.  and  are  sparingly  soluble  in  cold  (1-2454)  water, 


PROTEINS. 


7< 


but  much  more  so  in  boiling  (1-154)  water.     Tyrosine  forms  sol- 
uble salts  with  alkalis,  ammonia  or  mineral  acids,  and  is  soluble, 


Fig.  22. 


Fischer  Apparatus. 

Reproduced  from  a  photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University  of 
Pennsylvania.  The  negative  was  furnished  by  Dr.  T.  B.  Osborne,  of  New  Haven, 
Conn. 

A,  Tank  into  which  freezing  mixture  is  pumped  and  from  which  it  flows  through 
the  condenser,  B  ;  C,  flask  from  which  the  esters  are  distilled,  the  distillate  being 
collected  in  D  ;  E,  a  Dewar  flask  containing  liquid  air  serving  as  a  cooler  for  con- 
densing tube  F  ;  G  and  G' ,  tubes  leading  to  the  Geryck  pump  by  which  the  vacuum  is 
maintained  ;  /,  tube  leading  to  a  McLeod  gauge  (not  shown  in  figure)  ;  /,  a  bath  con- 
taining freezing  mixture  in  which  the  receiver  D  is  immersed ;  K,  a  bath  of  water 
during  the  first  part  of  the  distillation  and  of  oil  during  the  last  part  of  the  process  ; 
1-5,  stop  cocks  which  permit  the  cutting  out  of  different  parts  of  the  apparatus  as  the 
procedure  demands. 


with  difficulty,  in  acetic  acid.     It  responds  to  Millon's  reaction,  thus 
showing  the  presence  of  the  hydroxyphenyl  group,  but  gives  no 


72 


PHYSIOLOGICAL    CHEMISTRY. 


other  protein  test.  The  aromatic  groups  present  in  tyrosine,  phenyl- 
alanine and  tryptophane  cause  proteins  to  yield  a  positive  xantho- 
proteic reaction.     In  severe  cases  of  typhoid  fever  and  smallpox, 


Fig. 


23- 


Tyrosine. 

in  acute  yellow  atrophy  of  the  liver,  and  in  acute  phosphorus  poison- 
ing-, tyrosine  has  been  found  in  the  urine.  Tyrosine  crystals  are 
shown  in  Fig.  23,  above. 

Cystine,  C6H1204N2S2. — Friedmann  has  recently  shown  cystine 
to  be  the  disulphide  of  a-amino-fi-thiolactic  acid1  and  to  possess  the 
following  structural  formula: 

CH2-S — kvCxl2 

CHNH2    CH-NH2 

I  I 

COOH        COOH. 

Cystine  is  the  principal  sulphur-containing  body  obtained  from 
the  decomposition  of  protein  substances.  It  is  obtained  in  greatest 
amount  as  a  decomposition  product  of  such  keratin-containing  tis- 
sues as  horn,  hoof  and  hair.  Cystine  occurs  in  small  amount  in 
normal  urine  and  is  greatly  increased  in  quantity  under  certain 
pathological  conditions.  It  crystallizes  in  thin,  colorless  hexagonal 
plates  which  are  shown  in  Fig.  24,  p.  73.  Cystine  is  very  slightly 
soluble  in  water  but  its  salts,  with  both  bases  and  acids,  are  readily 
soluble  in  water.     It  is  lsevo-rotatory. 

1  Also  called   a-diamino-/3-dithio-dilactylic  acid. 


PROTEINS.  73 

It  has  recently  been  claimed  that  cystine  occurs  in  two  forms, 
i.  e.,  stone-cystine  and  protein-cystine  and  that  these  two  forms  are 
distinct  in  their  properties.     This  view  is  incorrect. 

Fig.  2a. 


Cystine. 

For  a  discussion  of  cystine  sediments  in  urine  see  Chapter  XX. 

Tryptophane,  CnH12N202. — According  to  Ellinger,  tryptophane 
is  indol-amino-propionic  acid.  Recently  Ellinger  and  Flamand  have 
shown  that  it  possesses  the  following  formula : 

/\ C-CH2CH(NH2)-COOH 

U\Ah 

NH 

Tryptophane  is  the  mother-substance  of  indole,  skatole,  skatole 
acetic  acid  and  skatole  carboxylic  acid,  all  of  which  are  formed  as 
secondary  decomposition  products  of  proteins.  Its  presence  in 
protein  substances  may  be  shown  by  means  of  the  Adamkiewicz 
reaction  or  the  Hopkins-Cole  reaction  (see  page  91).  It  may  be 
detected  in  a  tryptic  digestion  mixture  through  its  property  of  giving 
a  violet  color-reaction  with  bromine  water.  Tryptophane  is  yielded 
by  nearly  all  proteins  but  has  been  shown  to  be  entirely  absent  from 
zein,  the  prolamin  (alcohol-soluble  protein)  of  maize. 

Solutions  of  tryptophane  in  sodium  hydroxide  are  dextro-rotatory. 
Upon  being  heated  to  266 °  C.  tryptophane  decomposes  with  the  evo- 
lution of  gas. 


74 


PHYSIOLOGICAL    CHEMISTRY. 


Histidine,    C6H9N302. — Histidine    is    a-amino-fS-imidazol-pro- 
pionic  acid  with  the  following  structural  formula : 

H    NH2 

HC  =  C-C-C-COOH. 

I       I 
H    H 

HN\/N 

CH 

The  histidine  obtained  from  proteins  is  laevo-rotatory.     It  has 
been  obtained  from  all  the  proteins  thus  far  examined,  the  majority 
of    them  yielding  about  2.5  per  cent  of    the  amino  acid.     How- 
Fig.  25. 


Histidine  Dichloride. 

ever,  about  1 1  per  cent  was  obtained  by  Abderhalden  from  globin, 
the  protein  constituent  of  oxyhemoglobin  and  about  13  per  cent 
by  Kossel  and  Kutscher  from  the  protamine  sturine. 

Crystals  of  histidine  dichloride  are  shown  in  Fig.  25,  above. 

Knoop's  Color  Reaction  for  Histidine. — To  an  aqueous  solu- 
tion of  histidine  or  a  histidine  salt  in  a  test-tube  add  a  little  bromine 
water.  A  yellow  coloration  develops  in  the  cold  and  upon  further 
addition  of  bromine  water  becomes  permanent.  If  the  tube  be 
heated,1  the  color  will  disappear  and  will  shortly  be  replaced  by  a 
faint  red  coloration  which  gradually  passes  into  a  deep  wine  red. 
Usually  black,  amorphous  particles  separate  out  and  the  solution 
becomes  turbid. 

1  The  same  reaction  will  take  place  in  the  cold  more  slowly. 


PROTEINS.  75 

The  reaction  cannot  be  obtained  in  solutions  containing  free 
alkali.  It  is  best  to  use  such  an  amount  of  bromine  as  will  produce 
a  permanent  yellow  color  in  the  cold.  The  use  of  a  less  amount  of 
bromine  than  this  produces  a  weak  coloration  whereas  an  excess  of 
bromine  prevents  the  reaction.  The  test  is  not  very  delicate,  but 
a  characteristic  reaction  may  always  be  obtained  in  I  :  iooo  solu- 
tions. The  only  histidine  derivative  which  yields  a  similar  colora- 
tion is  imidazolethylamine,  and  the  reaction  in  this  case  is  rather 
weak  as  compared  with  the  color  obtained  with  histidine  or  histi- 
dine salts. 

Valine,  C5HnN02. — The  amino-valerianic  acid  obtained  from 
proteins  is  a-amino-isovalerianic  acid,  and  as  such  bears  the  follow- 
ing formula : 

CH3   NH2 

H-C C-COOH. 

I  I 

CH3   H 

It  closely  resembles  leucine  in  many  of  its  properties,  but  is  more 
soluble  in  water.  It  is  a  difficult  matter  to  identify  valine  in  the 
presence  of  leucine  and  isoleucine  inasmuch  as  these  amino  acids 
crystallize  together  in  such  a  way  that  the  combination  persists  even 
after  repeated  recrystallizations.     Valine  is  dextro-rotatory. 

Arginine,  C6H14N402. — Arginine  is  guanidine-a-amino-valeri- 
anic  acid  and  possesses  the  following  structural  formula : 

H   H    H   NH2 

NH-C-C-C-C-COOH. 

I         I      I       I       I 
NH  =  C        H   H    H   H 

NH2 

It  has  been  obtained  from  every  protein  so  far  subjected  to  decom- 
position. The  arginine  obtained  from  proteins  is  dextro-rotatory, 
and  has  pronounced  basic  properties,  reacts  strongly  alkaline  to 
litmus,  and  forms  stable  carbonates.  Because  of  these  facts,  some 
investigators  consider  arginine  to  be  the  nucleus  of  the  protein 
molecule.  It  is  obtained  in  widely  different  amounts  from  different 
proteins,  over  85  percent  of  certain  protamines  having  been  obtained 
in  the  form  of  this  amino  acid.     It  is  claimed  that  in  the  ordinary 


76 


PHYSIOLOGICAL    CHEMISTRY. 


metabolic  activities  of  the  animal  body  arginine  gives  rise  to  urea. 
While  this  claim  is  probably  true,  it  should,  at  the  same  time,  be 
borne  in  mind  that  the  greater  part  of  the  protein  nitrogen  is 
eliminated  as  urea  and  that,  therefore,  but  a  very  small  part  can 
arise  from  arginine. 

Leucine,  C6H13N02. — Leucine  is  an  abundant  end-product  of 
the  decomposition  of  protein  material,  and,  together  with  glycocoll, 
was  the  first  of  these  products  to  be  discovered  (1820).  It  is 
a-amino-isobutyl-acetic  acid,  and  therefore  has  the  following  for- 
mula : 


CIL 


NIL 


CH-CH.-C-COOH. 


CIL 


H 


The  leucine  which  results  from  protein  decomposition  is  /-leucine. 
Leucine  is  present  normally  in  the  pancreas,  thymus,  thyroid,  spleen, 
brain,  liver,  kidneys  and  salivary  glands.  It  has  been  found  patho- 
logically in  the  urine  (in  acute  yellow  atrophy  of  the  liver,  in  acute 
phosphorus  poisoning  and  in  severe  cases  of  typhoid  fever  and 
smallpox),  and  in  the  liver,  blood  and  pus. 


Fig.  26. 


Leucine. 


Pure  leucine  crystallizes  in  thin,  white  hexagonal  plates.  Crystals 
of  pure  leucine  are  reproduced  in  Fig.  26.  It  is  rather  easily  soluble 
in  water  (46  parts),  alkalis,  ammonia  and  acids.    On  rapid  heating 


PROTEINS.  77 

to  2950  C,  leucine  decomposes  with  the  formation  of  carbon 
dioxide,  ammonia  and  amylamine.  Aqueous  solutions  of  leucine 
obtained  from  proteins  are  lsevo-rotatory,  but  its  acid  or  alkaline, 
solutions  are  dextro-rotatory.  So-called  impure  leucine1  is  a  slightly 
refractive  substance,  which  generally  crystallizes  in  balls  having 
a  radial  structure  or  in  aggregations  of  spherical  bodies,  Fig.  104, 
Chapter  XX. 

Isoleucine,  CGH13N02. — Isoleucine  is  a-amino-methyl-ethyl-pro- 
pionic  acid,  and  possesses  the  following  structural  formula: 

CH3    NH2 
H  •  C-COOH. 


A 


2H5  H 

This  amino  acid  was  recently  discovered  by  Ehrlich.  Its  presence 
has  been  established  among  the  decomposition  products  of  only  a 
few  proteins  although  it  probably  occurs  among  those  of  many  or 
most  of  them.  Ehrlich  has  shown  that  the  d-amyl  alcohol  which 
is  produced  by  yeast  fermentation  originates  from  isoleucine  and 
the  isoamylalcohol  originates  frOm  leucine.  Isoleucine  is  dextro- 
rotatory. 

Lysine,  CGH14N202. — The  three  bodies,  lysine,  arginine  and  his- 
tidine,  are  frequently  classed  together  as  the  hexone  bases.  Lysine 
was  the  first  of  the  bases  discovered.  It  is  a-e-diamino-caproic  acid 
and  hence  possesses  the  following  structure : 

NH2H   H   H    NH2 

1    X   1    I    I 

H-C  -  C-C-C-C-COOH. 
H     H    H   H    H 

It  is  dextro-rotatory  and  is  found  in  relatively  large  amount  in 
casein  and  gelatin.  Lysine  is  obtained  from  nearly  all  proteins  but 
is  absent  from  the  vegetable  proteins  which  are  soluble  in  strong 
alcohol.  It  is  the  mother-substance  of  cadaverin  and  has  never 
been  obtained  in  crystalline  form.     Lysine  is  usually  obtained  as 

1  These  balls  of  so-called  impure  leucine  do  contain  considerable  leucine,  but 
inasmuch  as  they  may  contain  many  other  things  it  is  a  bad  practice  to  allude 
to  them  as  leucine. 


78 


PHYSIOLOGICAL    CHEMISTRY. 


the  picrate  which  is  sparingly  soluble  in  water   and  crystallizes 
readily.    These  crystals  are  shown  in  Fig.  27. 

Fig.  27. 


Lysine  Picrate. 


Aspartic  Acid,  C4H7N04. — Aspartic  acid  is  amino-succinic  acid 
and  has  the  following  structural  formula : 


Aspartic  Acid. 


PROTEINS. 


79 


The  amide  of  aspartic  acid,  asparaginic,  is  very  widely  distributed 
in  the  vegetable  kingdom.  The  crystalline  form  of  aspartic  acid 
is  exhibited  in  Fig.  28. 

Aspartic  acid  has  been  found  among  the  decomposition  products 
of  all  the  proteins  examined,  except  the  protamines.  It  has  not  been 
obtained,  however,  in  very  large  proportion  from  any  of  them.  The 
aspartic  acid  obtained  from  protein  is  laevo-rotatory. 

Glutamic  Acid,  C5H9N04. — This  acid  is  a-amino-normal-glntaric 
acid  and  as  such  bears  the  following  graphic  formula: 

NH2 

CHCOOH 

I 
CH2 

CH2COOH. 

Glutamic  acid  is  yielded  by  all  the  proteins  thus  far  examined, 
except  the  protamines,  and  by  most  of  these  in  larger  amount  than 
any  other  of  their  decomposition  products.  It  is  yielded  in  espe- 
cially large  proportion  by  most  of  the  proteins  of  seeds,  41.32  per 

Fig.  29. 


m      e 


Glutamic    Acid. 

Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University 

of   Pennsylvania. 

cent  having  been  obtained  very  recently  by  Kleinschmitt  from  the 
hydrolysis  of  hordcin  the  prolamin  of  barley.     This  is  the  largest 


8o 


PHYSIOLOGICAL    CHEMISTRY. 


amount  of  any  single  decomposition  product  yet  obtained  from  any 
protein  except  the  protamines.1 

Glutamic  acid  and  aspartic  acid  are  the  only  dibasic  acids  which 
have  thus  far  been  obtained  as  decomposition  products  of  proteins. 
As  there  is  an  apparent  relation  between  the  proportion  of  these 
acids  and  that  of  ammonia  which  the  different  proteins  yield  it  is 
possible  that  one  of  the  carboxyl  groups  of  these  acids  is  united  with 
NH2  as  an  amide,  the  other  carboxyl  group  being  united  in  poly- 
peptide union  (see  page  66)  with  some  other  amino  acid.  This 
might  be  represented  by  the  following  formula : 

R-CHNH-COOH 

C0-CHNH9-CH9-CH9-C0NH9. 


It  has  not  been  definitely  proven,  however,  that  this  form  of  link- 
ing actually  occurs. 

The  glutamic  acid,  yielded  by  proteins  upon  hydrolysis,  is  dextro- 
rotatory.  Crystals  of  glutamic  acid  are  reproduced  in  Fig.  29,  page  79. 

Proline,  C5H9N02. — Proline  is  a-pyrrolidine-carboxylic  acid  and 
possesses  the  following  graphic  structure : 

HoC       CHo 


H2C\/CHCOOH. 
NH 

Fig.  30. 


L.ffiVO-a-PROLINE. 

1Up  to  this  time  the  yield  of  37.33  per  cent  obtained  by  Osborne  and  Harris 
from  gliadin  of  wheat  was  the  maximum  yield 


PROTEINS.  8 1 

Proline  was  first  obtained  as  a  decomposition  product  of  casein. 
Proline  obtained  from  proteins  is  lsevo-rotatory  and  is  the  only 
protein  decomposition  product  which  is  readily  soluble  in  alcohol. 
It  is  also  one  of  the  few  heterocyclic  compounds  obtained  from  pro- 
teins. Proline  was  quite  recently  discovered  but  has  since  been 
found  among  the  decomposition  products  of  all  proteins  except  the 
protamines.  The  maximum  yield  reported  is  13.73  Per  cent  obtained 
by  Osborne  and  Clapp  from  the  hydrolysis  of  hordein.  The  crystal- 
line form  of  lav o-a- proline  is  shown  in  Fig-.   30,  and  the  copper 

Fig.  31. 


Copper  Salt  of  Proline. 

Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University 

of    Pennsylvania. 

salt  of  proline  is  represented  by  a  micro-photograph  in  Fig.  31, 
above.  The  crystals  of  the  copper  salt  have  a  deep  blue  color  but 
when  they  lose  their  water  of  crystallization  they  assume  a  char- 
acteristic violet  color. 

Oxyproline,  C5H9N03. — Oxyproline  was  recently  discovered  by 
Fischer.  It  has  as  yet  been  obtained  from  only  a  few  proteins,  but 
this  may  be  due  to  the  fact  that  only  a  few  have  been  examined  for 
its  presence.    Its  structure  has  not  yet  been  established. 

Diaminotrihydroxydodecanoic  Acid,  C12PI2gNo05. — This  amino 
acid  was  discovered  by  Fischer  and  Abderhalden  as  a  product  of 
the  hydrolysis  of  casein.  It  has  thus  far  been  obtained  from  no 
other  source.  It  is  lsevo-rotatory  and  its  constitution  has  not  been 
determined. 


82  physiological  chemistry. 

Experiments. 

While  the  ordinary  courses  in  physiological  chemistry  preclude 
any  extended  study  of  the  decomposition  products  of  proteins,  the 
manipulation  of  a  simple  decomposition  and  the  subsequent  isola- 
tion and  study  of  a  few  of  the  products  most  easily  and  quickly 
obtained  will  not  be  without  interest.1  To  this  end  the  student  may 
use  the  following  decomposition  procedure :  Treat  the  protein  in  a 
large  flask  with  water  containing  3-5  per  cent  of  H2S04  and  place 
it  on  a  water-bath  until  the  protein  material  has  been  decomposed 
and  there  remains  a  fine,  fluffy,  insoluble  residue.  Filter  off  this 
residue  and  neutralize  the  filtrate  with  Ba(OH)2  and  BaC03. 
Filter  off  the  precipitate  of  BaS04  which  forms  and  when  certain 
that  the  fluid  is  neutral  or  faintly  acid,2  concentrate  (first  on  a  wire 
gauze  and  later  on  a  water-bath)  to  a  syrup.  This  syrup  contains  the 
end-products  of  the  decomposition  of  the  protein,  among  which  are 
proteoses,  peptones,  tyrosine,  leucine,  etc.  Add  95  per  cent  alcohol 
slowly  to  the  warm  syrup  until  no  more  precipitate  forms,  stirring 
continuously  with  a  glass  rod.  This  precipitate  consists  of  proteoses 
and  peptones.  Gather  the  sticky  precipitate  on  the  rod  or  the  sides 
of  the  dish  and,  after  warming  the  solution  gently  for  a  few  mo- 
ments, filter  it  through  a  filter  paper  which  has  not  been  previously 
moistened.  After  dissolving  the  precipitate  of  proteoses  and  pep- 
tones in  water3  the  solution  may  be  treated  according  to  the  method 
of  separation  given  on  page  114. 

The  leucine  and  tyrosine,  etc.,  are  in  solution  in  the  warm  alcoholic 

filtrate.    Concentrate  this  filtrate  on  the  water-bath  to  a  thin  syrup, 

transfer  it  to  a  beaker,  and  allow  it  to  stand  over  night  in  a  cool 

place  for  crystallization.     The  tyrosine  first  crystallizes   (Fig.  23, 

page  72),  followed  later  by  the  formation  of  characteristic  crystals 

of  impure  leucine  (see  Fig.  105,  Chapter  XX).     After  examining 

these  crystals  under  the  microscope,  strain  off  the  crystalline  material 

through  fine  muslin,  heat  it  gently  in  a  little  water  to  dissolve  the  leu- 

1The  procedure  here  set  forth  has  nothing  in  common  with  the  procedure 
by  means  of  which  the  long  line  of  decomposition  products  just  enumerated  are 
obtained.  This  latter  process  is  an  exceedingly  complicated  one  which  is  entirely 
outside  the  province  of  any  course  in  physiological  chemistry. 

2  If  the  solution  is  alkaline  in  reaction  at  this  point,  the  amino  acids  will  be 
broken  down  and  ammonia  will  be  evolved. 

3  At  this  point  the  aqueous  solution  of  the  proteoses  and  peptones  may  be 
filtered  to  remove  any  BaS04  which  may  still  remain.  Tyrosine  crystals  will 
also  be  found  here,  since  it  is  less  soluble  than  the  leucine  and  may  adhere  to 
the  proteose-peptone  precipitate.  Add  the  crystals  of  tyrosine  to  the  warm  al- 
cohol filtrate. 


PROTEINS.  83 

cine  (the  tyrosine  will  be  practically  insoluble)  and  filter.  Concen- 
trate the  filtrate  and  allow  it  to  stand  in  a  cool  place  over  night  for 
the  crude  leucine  to  crystallize.  Filter  off  the  crystals  and  use  them 
in  the  tests  for  leucine  given  on  page  84.  The  crystals  of  tyrosine 
remaining  on  the  paper  from  the  first  filtration  may  be  used  in  the 
tests  for  tyrosine  as  given  below.  If  desired,  the  tyrosine  and  leucine 
may  be  purified  by  recrystallizing  in  the  usual  manner.  Habermann 
has  suggested  a  method  of  separating  leucine  and  tyrosine  by  means 
of  glacial  acetic  acid. 

Experiments  on  Tyrosine. 

Make  the  following  tests  with  the  tyrosine  crystals  already  pre- 
pared or  upon  some  pure  tyrosine  furnished  by  the  instructor. 

1.  Microscopical  Examination. — Place  a  minute  crystal  of  ty- 
rosine on  a  slide,  add  a  drop  of  water,  cover  with  a  cover  glass,  and 
examine  microscopically.  Now  run  more  water  under  the  cover 
glass  and  warm  in  a  bunsen  flame  until  the  tyrosine  has  dissolved. 
Allow  the  solution  to  cool  slowly  then  examine  again  microscopically 
and  compare  the  crystals  with  those  shown  in  Fig.  23,  page  72. 

2.  Solubility. — Try  the  solubility  of  very  small  amounts  of  tyro- 
sine in  cold  and  hot  water,  cold  and  hot  95  per  cent  alcohol,  dilute 
NH4OH,  dilute  KOH  and  dilute  HC1. 

3.  Sublimation. — Place  a  little  tyrosine  in  a  dry  test-tube,  heat 
gently  and  notice  that  the  material  does  not  sublime.  How  does  this 
compare  with  the  result  of  Experiment  3  under  Leucine? 

4.  Hoffman's  Reaction. — This  is  the  name  given  to  Millon's 
reaction  when  employed  to  detect  tyrosine.  Add  about  3  c.c.  of 
water  and  a  few  drops  of  Millon's  reagent  to  a  little  tyrosine  in  a 
test-tube.  Upon  dissolving  the  tyrosine  by  heat  the  solution  gradu- 
ally darkens  and  may  assume  a  dark  red  color.  What  group  does 
this  test  show  to  be  present  in  tyrosine? 

5.  Piria's  Test. — Warm  a  little  tyrosine  on  a  watch  glass  on  a 
boiling  water-bath  for  20  minutes  with  3-5  drops  of  cone.  H2S04. 
Tyrosine  sulphuric  acid  is  formed  in  the  process.  Cool  the  solution 
and  wash  it  into  a  small  beaker  with  water.  Now  add  CaC03  in 
substance  slowly  with  stirring,  until  the  reaction  of  the  solution  is 
no  longer  acid.  Filter,  concentrate  the  filtrate  and  add  to  it  a  few 
drops  (avoid  an  excess)  of  very  dilute  neutral  ferric  chloride.  A 
purple  or  violet  color,  due  to  the  formation  of  the  ferric  salt  of 
tyrosine-sulphuric  acid,  is  produced.  This  is  one  of  the  most  satis- 
factory tests  for  the  identification  of  tyrosine. 


84  PHYSIOLOGICAL    CHEMISTRY. 

6.  Morner's  Test. — Add  about  3  c.c.  of  Morner's  reagent1  to  a 
little  tyrosine  in  a  test-tube,  and  gently  raise  the  temperature  to  the 
boiling-point.    A  green  color  results. 

Experiments  on  Leucine. 

Make  the  following  tests  upon  the  leucine  crystals  already  pre- 
pared or  upon  some  pure  leucine  furnished  by  the  instructor. 

1,  2  and  3.  Repeat  these  experiments  according  to  the  directions 
given  under  Tyrosine  (page  83). 

1  Morner's  reagent  is  prepared  by  thoroughly  mixing  i   volume  of  formalin. 
45  volumes  of  distilled  water  and  55  volumes  of  concentrated  sulphuric  acid. 


CHAPTER    V. 

PROTEINS:    THEIR   CLASSIFICATION   AND 
PROPERTIES. 

From  what  has  already  been  said  in  Chapter  IV,  regarding  the 
protein  substances  it  will  be  recognized  that  the  grouping  of  the 
diverse  forms  of  this  class  of  substances  in  a  logical  manner  is  not 
an  easy  task.  The  fats  and  carbohydrates  may  be  classified  upon 
the  fundamental  principles  of  their  stereo-chemical  relationships 
whereas  such  a  system  of  classification  in  the  case  of  the  proteins 
is  absolutely  impossible  since,  as  we  have  already  stated,  the  mole- 
cular structure  of  these  complex  substances  is  unknown.  Because 
of  the  diversity  of  standpoint  from  which  the  proteins  may  be 
viewed,  relative  to  their  grouping  in  the  form  of  a  logically  classified 
series,  it  is  obvious  that  there  is  an  opportunity  for  the  presen- 
tation of  classifications  of  a  widely  divergent  character.  The  fact 
that  there  were  until  recently  at  least  a  dozen  different  classifica- 
tions which  were  recognized  by  various  groups  of  English-speaking 
investigators,  emphasizes  the  difficulties  in  the  way  of  the  individual 
or  individuals  who  would  offer  a  classification  which  should  merit 
universal  adoption.  Realizing  the  great  handicap  and  disadvantage 
which  the  great  diversity  of  the  protein  classifications  was  forcing 
upon  the  workers  in  this  field  the  British  Medical  Association  re- 
cently drafted  a  classification  which  appealed  to  that  body  of  scien- 
tists as  fulfilling  all  requirements  and  presented  it  for  the  consid- 
eration of  the  American  Physiological  Society  and  the  American 
Society  of  Biological  Chemists.  The  outcome  of  this  has  been  that 
there  are  now  only  two  protein  classifications  which  are  recognized 
by  English-speaking  scientists,  one  the  British  Classification  the  other 
the  American  Classification.  These  classifications  are  very  similar 
and  doubtless  will  ultimately  be  merged  into  a  single  classification. 
In  our  consideration  of  the  proteins  we  shall  conform  in  all  de- 
tails to  the  American  Classification.  In  this  connection  we  will  say, 
however,  that  we  feel  that  the  British  Medical  Association  has 
strong  grounds  for  preferring  the  use  of  the  term  scleroprotcins  for 
albuminoids  and  chromoproteins  for  haemoglobins.  The  two  classi- 
fications are  as  follows : 

85 


86  PHYSIOLOGICAL    CHEMISTRY. 

CLASSIFICATION    OF   PROTEINS   ADOPTED   BY 
THE  AMERICAN  PHYSIOLOGICAL  SOCIETY 
AND  THE  AMERICAN  SOCIETY  OF 
BIOLOGICAL  CHEMISTS. 

I.  SIMPLE    PROTEINS. 

Protein  substances  which  yield  only  a-amino  acids  or  their  de- 
rivatives on  hydrolysis. 

(a)  Albumins. — Soluble  in  pure  water  and  coagulable  by  heat, 
e.  g.,  ovalbumin,  serum  albumin,  lactalbumin,  vegetable  albumins. 

(b)  Globulins. — Insoluble  in  pure  water  but  soluble  in  neutral 
solutions  of  salts  of  strong  bases  with  strong  acids,1  e.  g.,  serum 
globulin,  ovo globulin,  edestin,  amandin  and  other  vegetable  globu- 
lins. 

(c)  Glutelins. — Simple  proteins  insoluble  in  all  neutral  solvents 
but  readily  soluble  in  very  dilute  acids  and  alkalis,2  e.  g.,  glutenin. 

(d)  Alcohol-soluble  proteins  (Prolamins).3 — Simple  proteins 
soluble  in  70-80  per  cent  alcohol,  insoluble  in  water,  absolute  alcohol 
and  other  neutral  solvents,4  e.  g.,  zein,  gliadin,  hordein  and  bynin. 

(e)  Albuminoids. — Simple  proteins  possessing  a  similar  struc- 
ture to  those  already  mentioned,  but  characterized  by  a  pronounced 
insolubility  in  all  neutral  solvents,5  e.  g.,  elastin,  collagen,  keratin. 

(/)  Histones. — Soluble  in  water  and  insoluble  in  very  dilute 
ammonia,  and,  in  the  absence  of  ammonium  salts,  insoluble  even  in 
excess  of  ammonia;  yield  precipitates  with  solutions  of  other  pro- 
teins and  a  coagulum  on  heating  which  is  easily  soluble  in  very 
dilute  acids.  On  hydrolysis  they  yield  a  large  number  of  amino 
acids  among  which  the  basic  ones  predominate.  In  short  histones 
are  basic  proteins  which  stand  between  protamines  and  true  pro- 
teins, e.  g.,  globin,  thymus  histone,  scombrone. 

1  The  precipitation  limits  with  ammonium  sulphate  should  not  be  made  a  basis 
for  distinguishing  the  albumins  from  the  globulins. 

2  Such  substances  occur  in  abundance  in  the  seeds  of  cereals  and  doubtless 
represent  a  well-defined  natural  group  of  simple  proteins. 

3  The  name  prolamins  has  been  suggested  for  these  alcohol-soluble  proteins 
by  Dr.  Thomas  B.  Osborne  (Science,  1908,  XXVIII,  p.  417).  It  is  a  very- 
fitting  term  inasmuch  as  upon  hydrolysis  they  yield  particularly  large  amounts 
of  proline  and  ammonia. 

4  The  subclasses  defined  (a,  b,  c,  d,)  are  exemplified  by  proteins  obtained  from 
both  plants  and  animals.  The  use  of  appropriate  prefixes  will  suffice  to  indicate 
the  origin  of  the  compounds,  e.  g.,  ovoglobulin,  lactalbumin,  etc. 

5  These  form  the  principal  organic  constituents  of  the  skeletal  structure  of 
animals  and  also  their  external  covering  and  its  appendages.  This  definition  does 
not  provide  for  gelatin  which  is,  however,  an  artificial  derivative  of  collagen. 


PROTEINS.  87 

{g)  Protamines. — Simpler  polypeptides  than  the  proteins  in- 
cluded in  the  preceding  groups.  They  are  soluble  in  water,  uncoag- 
ulable  by  heat,  have  the  property  of  precipitating  aqueous  solutions 
of  other  proteins,  possess  strong  basic  properties  and  form  stable 
salts  with  strong  mineral  acids.  They  yield  comparatively  few 
amino  acids,  among  which  the  basic  ones  predominate.  They  are 
the  simplest  natural  proteins,  e.  g.,  sahnine,  sturine,  clupeine,  scorn- 
brine. 

II.  CONJUGATED    PROTEINS. 

Substances  which  contain  the  protein  molecule  united  to  some 
other  molecule  or  molecules  otherwise  than  as  a  salt. 

(a)  Nucleoproteins. — Compounds  of  one  or  more  protein  mole- 
cules with  nucleic  acid,  e.  g.,  cytoglobulin,  nucle ohist one. 

(b)  Glycoproteins. — Compounds  of  the  protein  molecule  with  a 
substance  or  substances  containing  a  carbohydrate  group  other  than 
a  nucleic  acid,  e.  g.,  mucins  and  mucoids  {osseomucoid,  tendomu- 
coid,  ichthulin,  helicoprotein) . 

(c)  Phosphoproteins. — Compounds  of  the  protein  molecule  with 
some,  as  yet  undefined,  phosphorus-containing  substances  other 
than  a  nucleic  acid  or  lecithin,1  e.  g.,  caseinogen,  vitellin. 

{d)  Haemoglobins. — Compounds  of  the  protein  molecule  with 
hsematin,  or  some  similar  substance,  e.  g.,  hcemoglobin,  hcemocya- 
nin. 

{e)  Lecithoproteins. — Compounds  of  the  protein  molecule  with 
lecithins,  e.  g.,  lecithans,  phosphatides. 

III.  DERIVED    PROTEINS. 

I.  Primary  Protein  Derivatives. 
Derivatives  of  the  protein  molecule  apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alteration  of  the  pro- 
tein molecule. 

(a)  Proteans. — Insoluble  products  which  apparently  result  from 
the  incipient  action  of  water,  very  dilute  acids  or  enzymes,  e.  g., 
myosan,  edestan. 

(b)  Metaproteins. — Products  of  the  further  action  of  acids  and 
alkalis  whereby  the  molecule  is  so  far  altered  as  to  form  products 
soluble  in  very  weak  acids  and  alkalis  but  insoluble  in  neutral  fluids, 
e.  g.,  acid  metaprotein  {acid  albuminate) ,  alkali  mctaprotein  {alkali 
albuminate) . 

1  The  accumulated  chemical  evidence  distinctly  points  to  the  propriety  of  classi- 
fying the  phosphoproteins  as  conjugated  compounds,  i.  c,  they  are  possibly 
esters  of  some  phosphoric  acid  or  acids  and  protein. 


55  PHYSIOLOGICAL    CHEMISTRY. 

(c)  Coagulated  Proteins. — Insoluble  products  which  result 
from  (i)  the  action  of  heat  on  their  solutions,  or  (2)  the  action  of 
alcohol  on  the  protein. 

2.  Secondary  Protein  Derivatives.1 

Products  of  the  further  hydrolytic  cleavage  of  the  protein 
molecule. 

(a)  Proteoses. — Soluble  in  water,  non-coagulable  by  heat,  and 
precipitated  by  saturating  their  solutions  with  ammonium — or  zinc 
sulphate,2  e.  g.,  protoproteose,  deuteroproteose. 

(b)  Peptones. — Soluble  in  water,  non-coagulable  by  heat,  but 
not  precipitated  by  saturating  their  solutions  with  ammonium  sul- 
phate,3 e.  g.,  antipeptone,  ampho peptone. 

(c)  Peptides. — Definitely  characterized  combinations  of  two  or 
more  amino  acids,  the  carboxyl  group  of  one  being  united  with  the 
amino  group  of  the  other  with  the  elimination  of  a  molecule  of 
water,4  e.  g.,  dipeptides,  tripeptides,  tetrapeptides,  pentapeptides. 


CLASSIFICATION     OF     PROTEINS     ADOPTED     BY 
THE   BRITISH    MEDICAL   ASSOCIATION. 

I.  Simple  Proteins. 

1.  Protamines,  e.  g.,  salmine,  clupeine. 

2.  Histones,  e.  g.,  globin,  scombrone. 

3.  Albumins,  e.  g.,  ovalbumin,  serum  albumin,  vegetable  albu- 
mins. 

4.  Globulins,  e.  g.,  serum  globulin,  ovoglobulin,  vegetable  glob- 
ulins. 

5.  Glutelins,  e.  g.,  glutenin. 

6.  Alcohol-soluble  proteins,  e.  g.,  zein,  gliadin. 

7.  Scleroproteins,  e.  g.,  elastin,  keratin. 

8.  Phosphoproteins,  e.  g.,  caseinogen,  vitellin. 

1  The  term  secondary  hydrolytic  derivatives  is  used  because  the  formation  of 
the  primary  derivatives  usually  precedes  the  formation  of  these  secondary 
derivatives. 

2  As  thus  defined,  this  term  does  not  strictly  cover  all  the  protein  derivatives 
commonly  called  proteoses,  e.  g.,  heteroproteose  and  dysproteose. 

8  In  this  group  the  kyrines  may  be  included.  For  the  present  it  is  believed  that 
it  will  be  helpful  to  retain  this  term  as  defined,  reserving  the  expression  peptide 
for  the  simpler  compounds  of  definite  structure,  such  as  dipeptides,  etc. 

4  The  peptones  are  undoubtedly  peptides  or  mixtures  of  peptides,  the  latter 
term  being  at  present  used  to  designate  those  of  definite  structure. 


PROTEINS.  89 

II.  Conjugated    Proteins. 

1.  Oncoproteins,  e.  g.,  mucins,  mucoids. 

2.  Nucleoproteins,  e.  g.,  nucleohistone,  cytoglobulin. 

3.  Chromoproteins,  e.  g.,  hemoglobin,  hccmocyanin. 

III.  Products  of  Protein  Hydrolysis. 

1.  Infraproteins,  e.  g.,  acid  infraprotein  (acid  albuminate) ,  alkali 
infraprotein  (alkali  albuminate). 

2.  Proteoses,  e.  g.,  protoproteose,  heteroproteose,  deuteroproteose. 

3.  Peptones,  e.  g.,  ainphopeptone,  antipeptone. 

4.  Polypeptides,  e.  g.,  dipeptides,  tripeptides,  tetrapeptides. 


CONSIDERATIONS   OF  THE  VARIOUS   CLASSES 
OF    PROTEINS. 

SIMPLE  PROTEINS. 

The  simple  proteins  are  true  protein  substances  which,  upon  hy- 
drolysis, yield  only  a-amino  acids  or  their  derivatives.  "Although 
no  means  are  at  present  available  whereby  the  chemical  individual- 
ity of  any  protein  can  be  established,  a  number  of  simple  proteins 
have  been  isolated  from  animal  and  vegetable  tissues  which  have 
been  so  well  characterized  by  constancy  of  ultimate  composition 
and  uniformity  of  physical  properties  that  they  may  be  treated  as 
chemical  individuals  until  further  knowledge  makes  it  possible  to 
characterize  them  more  definitely."  Under  simple  proteins  we  may 
class,  albumins,  globulins,  glutelins,  prolamins,  albuminoids,  his- 
tones  and  protamines. 

ALBUMINS. 

Albumins  constitute  the  first  class  of  simple  proteins  and  may 
be  defined  as  simple  proteins  which  are  coagulable  by  heat  and 
soluble  in  pure  (salt-free)  water.  Those  of  animal  origin  are  not 
precipitated  upon  saturating  their  neutral  solutions  at  300  C.  with 
sodium  chloride  or  magnesium  sulphate,  but  if  a  saturated  solution 
of  this  character  be  acidified  with  acetic  acid  the  albumin  precipi- 
tates. All  albumins  of  animal  origin  may  be  precipitated  by  sat- 
urating their  solutions  with  ammonium  sulphate.1     They  may  be 

1  In  this  connection,  Osborne's  observation  that  there  are  certain  vegetable 
albumins  which  are  precipitated  by  saturating  their  solutions  with  sodium  chlor- 
ide or  magnesium  sulphate  or  by  half-saturating  with  ammonium  sulphate,  is 
of   interest. 


90  PHYSIOLOGICAL    CHEMISTRY. 

thrown  out  of  solution  by  the  addition  of  a  sufficient  quantity  of 
a  mineral  acid,  whereas  a  weak  acidity  produces  a  slight  precipitate 
which  dissolves  upon  agitating  the  solution.  Metallic  salts  also 
possess  the  property  of  precipitating  albumins,  some  of  the  precipi- 
tates being  soluble  in  excess  of  the  reagent  whereas  others  are  in- 
soluble in  such  an  excess.  Of  those  proteins  which  occur  native 
the  albumins  contain  the  highest  percentage  of  sulphur,  ranging 
from  1.6  to  2.5  per  cent.  Some  albumins  have  been  obtained  in 
crystalline  form,  notably  egg  albumin,  serum  albumin  and  lactal- 
bumin  but  the  fact  that  they  may  be  obtained  in  crystalline  form 
does  not  necessarily  prove  them  to  be  chemical  individuals. 


GENERAL  COLOR  REACTIONS  OF  PROTEINS. 

These  color  reactions  are  due  to  a  reaction  between  some  one  or 
more  of  the  constituent  radicals  or  groups  of  the  complex  protein 
molecule  and  the  chemical  reagent  or  reagents  used  in  any  given 
test.  Not  all  proteins  contain  the  same  groups  and  for  this  reason 
the  various  color  tests  will  yield  reactions  varying  in  intensity  of 
color  according  to  the  nature  of  the  groups  contained  in  the  par- 
ticular protein  under  examination.  Various  substances  not  pro- 
teins respond  to  certain  of  these  color  reactions  and  it  is  therefore 
essential  to  submit  the  material  under  examination  to  several  tests 
before  concluding  definitely  regarding  its  nature. 

TECHNIQUE  OF  THE  COLOR  REACTIONS. 

i.  Millon's  Reaction. — To  5  c.c.  of  a  dilute  solution  of  egg 
albumin  in  a  test-tube  add  a  few  drops  of  Millon's  reagent.  A 
white  precipitate  forms  which  turns  red  when  heated.  This  test 
is  a  particularly  satisfactory  one  for  use  on  solid  proteins,  in 
which  case  the  reagent  is  added  directly  to  the  solid  substance  and 
heat  applied,  which  causes  the  substance  to  assume  a  red  color. 
Such  proteins  as  are  not  precipitated  by  mineral  acids,  for  example 
certain  of  the  proteoses  and  peptones,  yield  a  red  solution  instead 
of  a  red  precipitate. 

The  reaction  is  due  to  the  presence  of  the  hydroxy-phenyl  group, 
—  C6H4OH,  in  the  protein  molecule  and  certain  non-proteins  such 
as  tyrosine,  phenol  (carbolic  acid)  and  thymol  also  respond  to 
the  reaction.  Inasmuch  as  the  tyrosine  grouping  is  the  only  hy- 
droxy-phenyl  grouping  which   has   definitely  been   proven   to  be 


PROTEINS.  91 

present  in  the  protein  molecule  it  is  evident  that  protein  substances 
respond  to  Millon's  reaction  because  of  the  presence  of  this  tyro- 
sine complex.  The  test  is  not  a  very  satisfactory  one  for  use  in 
solutions  containing  inorganic  salts  in  large  amount,  since  the  mer- 
cury of  the  Millon's  reagent1  is  thus  precipitated  and  the  reagent 
rendered  inert.  This  reagent  is  therefore  never  used  for  the  detec- 
tion of  protein  material  in  the  urine. 

2.  Xanthoproteic  Reaction. — To  2-3  c.c.  of  egg  albumin  solu- 
tion in  a  test-tube  add  concentrated  nitric  acid.  A  white  precipi- 
tate forms,  which  upon  heating  turns  yellow  and  finally  dissolves, 
imparting  to  the  solution  a  yellow  color.  Cool  the  solution  and 
carefully  add  ammonium  hydroxide,  potassium  hydroxide  or  sod- 
ium hydroxide  in  excess.  Note  that  the  yellow  color  deepens  into 
an  orange.  This  reaction  is  due  to  the  presence  in  the  protein 
molecule  of  the  phenyl  group,  with  which  the  nitric  acid  forms 
certain  nitro  modifications.  The  particular  complexes  of  the  pro- 
tein molecule  which  are  of  especial  importance  in  this  connection  are 
those  of  tyrosine,  phenylalanine  and  tryptophane.  The  test  is  not  a 
satisfactory  one  for  use  in  urinary  examination  because  of  the 
color  of  the  end-reaction. 

3.  Adamkiewicz  Reaction. — Thoroughly  mix  1  volume  of  con- 
centrated sulphuric  acid  and  2  volumes  of  acetic  acid  in  a  test-tube, 
add  a  few  drops  of  egg  albumin  solution  and  heat  gently.  A 
reddish-violet  color  is  produced.  Gelatin  does  not  respond  to  this 
test.  This  reaction  shows  the  presence  of  the  tryptophane  group 
(see  next  experiment).  The  test  depends  upon  the  presence  of 
glyoxylic  acid,  CHO  ■  COOH  +  H20  or  CH(OH)2COOH, 
in  the  reagents.  This  is  shown  by  the  failure  to  secure  a 
positive  reaction  when  acetic  acid  free  from  glyoxylic  acid  is  used. 

Rosenheim  has  recently  advanced  the  view  that  the  reaction  may 
be  due  to  the  presence  of  oxidizing  agents  such  as  nitrous  acid 
and  ferric  salts  in  the  sulphuric  acid. 

4.  Hopkins-Cole  Reaction. — Place  1-2  c.c.  of  egg  albumin  solu- 
tion and  3  c.c.  of  glyoxylic  acid,  CHO "  COOH  -j-  H20  or 
CH(OH)2COOH,  solution  (Hopkins-Cole  reagent2)  in  a  test-tube 

1  Millon's  reagent  consists  of  mercury  dissolved  in  nitric  acid  containing  some 
nitrous  acid.  It  is  prepared  by  digesting  one  part  (by  weight)  of  mercury 
with  two  parts  (by  weight)  of  HN03  (sp.  gr.  1.42)  and  diluting  the  resulting 
solution  with  two  volumes  of  water. 

2  Hopkins-Cole  reagent  is  prepared  as  follows:  To  one  liter  of  a  saturated 
solution  of  oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  the  mixture 
to  stand  until  the  evolution  of  gas  ceases.  Filter  and  dilute  with  2-3  volumes 
of  water. 


92  PHYSIOLOGICAL    CHEMISTRY. 

and  mix  thoroughly.  In  a  second  tube  place  5  c.c.  of  concentrated 
sulphuric  acid.  Incline  the  tube  containing  the  sulphuric  acid 
and  by  means  of  a  pipette  allow  the  albumin-glyoxylic  acid 
solution  to  flow  carefully  down  the  side.  When  stratified  in  this 
manner  a  reddish-violet  color  forms  at  the  zone  of  contact  of  the 
two  fluids.  This  color  is  due  to  the  presence  of  the  tryptophane 
group.  Gelatin  does  not  respond  to  this  test.  For  formula  for 
tryptophane  see  page  73. 

5.  Biuret  Test. — To  2-3  c.c.  of  egg  albumin  solution  in  a  test- 
tube  add  an  equal  volume  of  concentrated  potassium  hydroxide  solu- 
tion, mix  thoroughly,  and  add  slowly  a  very  dilute  (2-5  drops  in  a 
test-tube  of  water)  cupric  sulphate  solution  until  a  purplish-violet  or 
pinkish-violet  color  is  produced.  The  depth  of  the  color  depends 
upon  the  nature  of  the  protein,  proteoses  and  peptones  giving  a  de- 
cided pink,  while  the  color  produced  with  gelatin  is  not  far  removed 
from  a  blue.  This  reaction  is  given  by  those  substances  which  con- 
tain two  amino  groups  in  their  molecule,  these  groups  either  being 
joined  directly  together  or  through  a  single  atom  of  nitrogen  or 
carbon.  The  amino  groups  mentioned  must  either  be  two  CONH2 
groups  or  one  CONH2  group  and  one  CSNH2,  C(NH)NH2  or 
CH2NH2  group.  It  follows  from  this  fact  that  substances  which 
are  non-protein  in  character  but  which  contain  the  necessary  groups 
will  respond  to  the  biuret  test.  As  examples  of  such  substances 
may  be  cited  oxamide, 

CONH2 


and  biuret, 


CONH2 

CONH2 

\ 
NH  . 

/ 
CONH2 

The  test  derives  its  name  from  the  fact  that  this  latter  sub- 
stance which  is  formed  on  heating  urea  to  1800  C.  (see  page  269), 
will  respond  to  the  test.  Protein  material  responds  positively  since 
there  are  two  CONH2  groups  in  the  protein  molecule. 

According  to  Schiff  the  end-reaction  of  the  biuret  test  is  depend- 
ent upon  the  formation  of  a  copper-potassium-biuret  compound 
(cupri-potassium  biuret  or  biuret  potassium  cupric  hydroxide). 
This  substance  was  obtained  by  Schiff  in  the  form  of  long  red 
needles.     It  has  the  following  formula  : 


PROTEINS.  93 

OH  OH 

CO  •  NH2 Cu NH2C0 

\      "  / 

NH  HN 

/  \ 

00  •  NH,— K      K— NH2  •  CO 

I  I 

OH  OH 

6.  Posner's  Modification  of  the  Biuret  Test. — This  test  is  par- 
ticularly satisfactory  for  use  on  dilute  protein  solutions,  and  is 
carried  out  as  follows :  To  some  dilute  egg  albumin  in  a  test- 
tube  add  one-half  its  volume  of  potassium  hydroxide  solution. 
Now  hold  the  tube  in  an  inclined  position  and  allow  some  very 
dilute  cupric  sulphate  solution,  made  as  suggested  on  page  92  (5), 
to  flow  down  the  side,  being  especially  careful  to  prevent  the  fluids 
from  mixing.  At  the  juncture  of  the  two  solutions  the  typical  end- 
reaction  of  the  biuret  test  should  appear  as  a  colored  zone  (see 
Biuret  Test,  page  92). 

7.  Liebermann's  Reaction. — Add  about  10  drops  of  concen- 
trated egg  albumin  solution  (or  a  little  dry  egg  albumin)  to  about 
5  c.c.  of  concentrated  HC1  in  a  test-tube.  Boil  the  mixture 
until  a  pinkish-violet  color  results.  This  color  was  originally  sup- 
posed to  indicate  the  presence  of  a  carbohydrate  group  in  the  pro- 
tein molecule,  the  furfurol  formed  through  the  action  of  the  acid 
upon  the  protein  reacting  with  the  hydroxy-phenyl  group  of  the  pro- 
tein producing  the  pinkish-violet  color.  It  is  now  considered  un- 
certain whether  the  carbohydrate  group  enters  into  the  reaction. 
Cole  has  called  attention  to  the  fact  that  a  blue  color  results  if  pro- 
tein material  which  has  been  boiled  with  alcohol  and  subsequently 
washed  with  ether  be  used  in  making  the  test.  He  believes  the  blue* 
color  to  be  due  to  an  interaction  between  the  glyoxylic  acid,  which 
was  present  as  an  impurity  in  the  ether  used  in  washing  the  protein, 
and  the  tryptophane  group  of  the  protein  molecule  which  was  split 
off  through  the  action  of  the  acid. 

8.  Acree-Rosenheim  Formaldehyde  Reaction. — Add  a  few 
drops  of  a  dilute  (1  15000)  solution  of  formaldehyde  to  2-3  c.c.  of 
egg  albumin  solution  in  a  test-tube.  Mix  thoroughly  and  after 
2-3  minutes  carefully  introduce  a  little  concentrated  sulphuric  acid 
into  the  tube  in  such  a  manner  that  the  two  solutions  do  not  mix. 
A  violet  zone  will  be  observed  at  the  point   of  juncture  of  the 


94  PHYSIOLOGICAL    CHEMISTRY. 

two  solutions  especially  if  the  mixture  is  slightly  agitated.  This 
color  probably  results  through  the  union  of  the  protein  and  the 
formaldehyde.  If  the  sulphuric  acid  is  added  to  the  protein  before 
the  formaldehyde  is  added  the  typical  end-reaction  is  not 
obtained.  So  far  as  is  known  this  is  a  specific  test  for  pro- 
teins. The  reaction  cannot  be  applied  satisfactorily  with  concen- 
trated formaldehyde. 

Rosenheim  claims  the  reaction  is  due  to  the  presence  of  oxidizing 
material  in  the  sulphuric  acid  and  that  when  pure  sulphuric  acid 
is  used  no  reaction  is  obtained.  He  advises  the  use  of  a  slight 
amount  of  an  oxidizing  agent,  e.  g.,  ferric  chloride  or  potassium 
nitrite  (0.005  gram  per  100  c.c.  of  sulphuric  acid)  in  order  to 
facilitate  the  reaction.  Rosenheim  further  states  that  proteins 
respond  to  the  formaldehyde  reaction  because  of  the  presence  of  the 
tryptophane  group,  a  statement  which  Acree  does  not  accept  as 
proven. 

9.  Bardach's  Reaction.1 — This  is  the  most  recent  test  which 
has  been  described  for  the  detection  of  protein  material.  The 
test  depends  upon  the  property  possessed  by  protein  substances  of 
preventing  the  formation  of  typical  iodoform  crystals  through  the 
interaction  of  an  alkaline  acetone  solution  with  iodopotassium 
iodide.  Instead  of  the  typical  hexagonal  plates  or  stellar  forma- 
tions of  iodoform  there  are  produced,  under  the  conditions  of  the 
test,  fine  yellow  needles  which  are  apparently  some  iodine  compound 
other  than  iodoform.  The  technique  of  the  test  is  as  follows : 
Place  about  5  c.c.  of  the  protein  solution2  under  examination  in 
a  test-tube,  add  2-3  drops  of  a  0.5  per  cent  solution  of  acetone  and 
sufficient  Lugol's  solution3  to  supply  a  moderate  excess  of  iodine 
and  produce  a  red-brown  coloration.  (The  amount  of  Lugol's  solu- 
tion necessary  will  depend  upon  the  content  of  protein,  sugar  and 
other  iodine-reacting  substances  in  the  solution  under  examination 
and  may  vary  from  one  drop  to  several  cubic  centimeters.)  Add 
an  excess  (ordinarily  about  3  c.c.)  of  concentrated  ammonium 
hydroxide  and  thoroughly  mix  the  solution.  Place  the  tube  in  the 
test-tube  rack,  examine  the  contents  at  intervals  of  five  minutes, 
and  when  it  is  evident  that  crystals  have  formed,  place  a  drop  of 

1  Bardach :  Zeitschrift  fur  Physiologische  Chemie,  1908,  LIV,  p.  355 ;  also 
Seaman  and  Gies:  Proceedings  of  the  Society  for  Experimental  Biology  and 
Medicine,  1908,  V,  p.  125. 

2  The  solution  should  not  contain  more  than  5  per  cent  of  protein  material. 

8  Dissolve  4  grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  of  dis- 
tilled water. 


PROTEINS.  95 

the  mixture  upon  a  microscopic  slide,  put  a  cover  glass  in  position 
and  examine  the  mixture  under  the  microscope.  The  formation 
of  canary  yellow  crystals  indicates  the  presence  of  protein  material 
in  the  solution  examined.  The  crystals  are  ordinarily  needle-like 
in  appearance  and  show  a  tendency  to  assume  rosette  or  bundle-like 
formations  but  under  certain  conditions  they  may  show  knobbed 
(nail-like)   and  branching  variations. 

If  a  moderate  excess  of  iodine  is  used  in  making  the  test  a  black 
precipitate  of  iodonitro  compounds  is  at  once  formed  upon  the  ad- 
dition of  the  ammonium  hydroxide  and  yellow  needles  are  sub- 
sequently deposited  upon  it.  In  case  just  the  proper  amount  of 
iodine  is  used,  the  solution  soon  assumes  a  yellow  color  and  the 
black  precipitate  formed  upon  the  addition  of  the  ammonium  hy- 
droxide is  gradually  transformed  more  or  less  completely  into  the 
yellow  crystals.  In  either  case  the  needles  ordinarily  form 
within  an  hour,  and  frequently  in  a  much  shorter  time.  If 
too  great  an  excess  of  iodine  is  employed  the  heavy  black  pre- 
cipitate may  obscure  or  even  prevent  the  reaction.  The  presence 
of  insufficient  iodine  or  excess  protein  may  likewise  prevent 
the  reaction.  In  tests  in  which  a  concentrated  protein  solution 
and  an  excess  of  iodine  are  used,  the  addition  of  ammonium 
hydroxide  immediately  produces  a  grayish-green  precipitate.  In 
such  instances,  if  the  proportions  are  favorable,  and  the  mixture  be 
stirred  with  a  glass  rod  for  a  few  minutes,  the  precipitate  is  grad- 
ually transformed  into  the  crystals  before  mentioned. 

It  is  probable  that  all  soluble  proteins  will  respond  to  Bardach's 
reaction,  but  the  relative  delicacy  of  the  reaction  as  well  as  the  value 
of  the  test  as  compared  with  other  protein  tests  remain  to  be  deter- 
mined. The  only  disturbing  factor  noted  thus  far  is  the  presence  of 
earthy  phosphates  in  the  solution  under  examination. 


PRECIPITATION  REACTIONS  AND  OTHER 
PROTEIN  TESTS. 

There  are  three  forms  in  which  proteins  may  be  precipitated 
i.  e.,  unaltered,  as  an  albuminate,  and  as  an  insoluble  salt.  An 
instance  of  the  precipitation  in  a  native  or  unaltered  condition  is 
seen  in  the  so-called  salting-out  experiments.  Various  salts,  notably 
(NH4)2S04,  ZnS04,  MgS04,Na2S04  and  NaCl  possess  the  power 
when  added  in  solid  form  to  certain  definite  protein  solutions,  of 


g6  PHYSIOLOGICAL    CHEMISTRY. 

rendering  the  menstruum  incapable  of  holding  the  protein  in  solu- 
tion, thereby  causing  the  protein  to  be  precipitated  or  salted-out 
to  use  the  common  term.  Mineral  acids  and  alcohol  also  precipitate 
proteins  unaltered.  Proteins  are  precipitated  as  albuminates  when 
treated  with  certain  metallic  salts,  and  precipitated  as  insoluble 
salts  when  weak  organic  acids  such  as  certain  of  the  alkaloidal 
reagents  are  added  to  their  solutions. 

It  is  generally  stated  that  globulins  are  precipitated  from  their 
solutions  upon  half  saturation  with  ammonium  sulphate  and  that  al- 
bumins are  precipitated  upon  complete  saturation  by  this  salt.  Com- 
paratively few  exceptions  were  found  to  this  rule  until  proteins 
of  vegetable  origin  came  to  be  more  extensively  studied.  These 
studies,  furthered  especially  by  Osborne,  and  associates,  have  dem- 
onstrated very  clearly  that  the  characterization  of  a  globulin  as  a 
protein  which  is  precipitated  by  half  saturation  with  ammonium 
sulphate,  can  no  longer  hold.  Certain  vegetable  globulins  have 
been  isolated  which  are  not  precipitated  by  this  salt  until  a  concen- 
tration is  reached  greater  than  that  secured  by  ]ialf -saturation. 
As  an  example  of  an  albumin  which  does  not  conform  to  the  defini- 
tion of  an  albumin  as  regards  its  precipitation  by  ammonium  sul- 
phate, may  be  mentioned  the  leucosin  of  the  wheat  germ  which  is 
precipitated  from  its  solution  upon  /^//-saturation  with  ammonium 
sulphate.  The  limits  of  precipitation  by  ammonium  sulphate, 
therefore,  do  not  furnish  a  sufficiently  accurate  basis  for  the 
differentiation  of  globulins  from  albumins.  It  has  further  been 
determined  that  a  given  protein  which  is  precipitable  by  ammonium 
sulphate  cannot  be  "  salted-out  "  by  the  same  concentration  of  the 
salt  under  all  conditions. 

Experiments. 

i.  Influence  of  Concentrated  Mineral  Acids,  Alkalis  and  Or- 
ganic Acids. — Prepare  five  test-tubes  each  containing  5  c.c.  of  con- 
centrated egg  albumin  solution.  To  the  first  add  concentrated 
H2S04,  drop  by  drop,  until  an  excess  of  the  acid  has  been  added. 
Note  any  changes  which  may  occur  in  the  solution.  Allow  the 
tube  to  stand  for  24  hours  and  at  the  end  of  that  period  observe 
any  alteration  which  may  have  taken  place.  Heat  the  tube  and 
note  any  further  change  which  may  occur.  Repeat  the  experiment 
in  the  four  remaining  tubes  with  concentrated  hydrochloric 
acid,  concentrated  nitric  acid,  concentrated  potassium  hydrox- 
ide and  acetic  acid.    How  do  strong  mineral  acids,  strong  alkalis  and 


PROTEINS.  97 

strong  organic  acids  differ  in  their  action  toward  protein  solutions? 

2.  Precipitation  by  Metallic  Salts. — Prepare  four  tubes  each 
containing  2-3  c.c.  of  dilute  egg  albumin  solution.  To  the  first 
add  mercuric  chloride,  drop  by  drop,  until  an  excess  of  the  reagent 
has  been  added,  noting  any  changes  which  may  occur.  Repeat  the 
experiment  with  plumbic  acetate,  argentic  nitrate,  cupric  sulphate, 
ferric  chloride  and  barium  chloride. 

Egg  albumin  is  used  as  an  antidote  for  lead  or  mercury  poisoning. 
Why? 

3.  Precipitation  by  Alkaloidal  Reagents. — Prepare  six  tubes 
each  containing  2-3  c.c.  of  dilute  egg  albumin  solution.  To  the  first 
add  picric  acid  drop  by  drop  until  an  excess  of  the  reagent  has  been 
added,  noting  any  changes  which  may  occur.  Repeat  the  experi- 
ment with  trichloracetic  acid,  tannic  acid,  phosphotungstic  acid, 
phospho-molybdic  acid  and  potassio-mercuric  iodide.  Acidify  with 
hydrochloric  acid  before  testing  with  the  three  last  reagents. 

4.  Heller's  Ring  Test. — Place  5  c.c.  of  concentrated  nitric  acid 
in  a  test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the 
dilute  albumin  solution  to  flow  slowly  down  the  side.  The  liquids 
should  stratify  with  the  formation  of  a  white  zone  of  precipitated 
albumin  at  the  point  of  juncture.  This  is  a  very  delicate  test  and 
is  further  discussed  on  p.  314. 

An  apparatus  called  the  albumoscope  or  horismacope  has  been 
devised  for  use  in  the  tests  of  this  character  and  has  met  with  con- 
siderable favor.  The  method  of  using  the  albumoscope  is  described 
below. 

Use  of  the  Albumoscope. — This  instrument  is  intended  to  facili- 
tate the  making  of  "  ring  "  tests  such  as  Heller's  and  Roberts'.  In 
making  a  test  about  5  c.c.  of  the  solution  under  examination  is  first 
introduced  into  the  apparatus  through  the  larger  arm  and  the  re- 
agent used  in  the  particular  test  is  then  introduced  through  the 
capillary  arm  and  allowed  to  flow  down  underneath  the  solution 
under  examination.  If  a  reasonable  amount  of  care  is  taken  there 
is  no  possibility  of  mixing  the  two  solutions  and  a  definitely  defined 
white  "  ring  "  is  easily  obtained  at  the  zone  of  contact. 

5.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent1  in  a 
test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  al- 
bumin solution  to  flow  slowly  down  the  side.     The  liquids  should 

1  Roberts'  reagent  is  composed  of  1  volume  of  concentrated  HNOs  and  5 
volumes  of  a  saturated  solution  of  MgSOi. 


98  PHYSIOLOGICAL    CHEMISTRY. 

stratify  with  the  formation  of  a  white  zone  of  precipitated  albumin 
at  the  point  of  juncture.  This  test  is  a  modification  of  Heller's 
ring  test  and  is  rather  more  satisfactory.  The  albumoscope  may 
also  be  used  in  making  this  test.     (See  page  97.) 

6.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent1 
in  a  test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow 
5  c.c.  of  albumin  solution,  acidified  with  acetic  acid,  to  flow  slowly 
down  the  side.  A  white  zone  will  form  at  the  point  of  contact. 
This  is  an  exceedingly  delicate  test,  in  fact,  too  delicate  for  ordi- 
nary clinical  purposes,  since  it  serves  to  detect  albumin  when  present 
in  the  merest  trace  (1  1250,000).  This  test  is  further  discussed  on 
page  316. 

7.  Jolles'  Reaction. — Shake  5  c.c.  of  albumin  solution  with  1 
c.c.  of  30  per  cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent2  in  a 
test-tube.  A  white  precipitate  of  albumin  should  form.  Care 
should  be  taken  to  use  the  correct  amount  of  acetic  acid.  For 
further  discussion  of  the  test  see  page  316. 

8.  Tanret's  Test. — To  5  c.c.  of  albumin  solution  in  a  test-tube 
add  Tanret's  reagent,3  drop  by  drop,  until  a  turbidity  or  precipitate 
forms.  This  is  an  exceedingly  delicate  test.  Sometimes  the  al- 
bumin solution  is  stratified  upon  the  reagent  as  in  Heller's  or  Rob- 
erts' ring  teste.  In  urine  examination  it  is  claimed  by  Repiton  that 
the  presence  of  urates  lowers  the  delicacy  of  the  test.  Tanret  has 
however  very  recently  made  a  statement  to  the  effect  that  the  re- 
moval of  urates  is  not  necessary  inasmuch  as  the  urate  precipitate 
will  disappear  on  warming  and  the  albumin  precipitate  will  not.  He 
says,  however,  that  mucin  interferes  with  the  delicacy  of  his  test 
and  should  be  removed  by  acidification  with  acetic  acid  and  filtration 
before  testing  for  albumin. 

1  Spiegler's  reagent  has  the  following  composition : 

Tartaric  acid 20  grams. 

Mercuric    chloride 40       " 

Glycerol   100       " 

Distilled    water 1000       " 

a  Jolles'' reagent  has  the  following  composition: 

Succinic    acid 40  grams. 

Mercuric    chloride 20       " 

Sodium    chloride 20       " 

Distilled    water 1000       " 

'Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  gram  of  mercuric 
chloride  in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide 
dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c.  with 
water  and  add  20  c.c.  of  glacial  acetic  acid  to  the  combined  solutions. 


PROTEINS.  99 

9.  Sodium  Chloride  and  Acetic  Acid  Test. — Mix  two  volumes 
of  albumin  solution  and  1  volume  of  a  saturated  solution  of  sodium 
chloride  in  a  test-tube,  acidify  with  acetic  acid  and  heat  to  b< tiling. 
The  production  of  a  cloudiness  or  the  formation  of  a  precipitate 
indicates  the  presence  of  albumin. 

10.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c. 
of  dilute  egg  albumin  solution  in  a  test-tube  add  5-10  drops  of  acetic 
acid.  Mix  well,  and  add  potassium  ferrocyanide,  drop  by  drop, 
until  a  precipitate  forms.     This  test  is  very  delicate. 

Schmiedl  claims  that  a  precipitate  of  Fe(Cn)6K2Zn  or  Fe(Cn)6- 
Zn2,  is  formed  when  solutions  containing  zinc  are  subjected  to  this 
test,  and  that  this  precipitate  resembles  the  precipitate  secured  with 
protein  solutions.  In  the  case  of  human  urine  a  reaction  was 
obtained  when  0.000022  gram  of  zinc  per  cubic  centimeter  was  pres- 
ent. Schmiedl  further  found  that  the  urine  collected  from  rabbits 
housed  in  zinc-lined  cages  possessed  a  zinc  content  which  was  suffi- 
cient to  yield  a  ready  response  to  the  test.  Zinc  is  the  only  in- 
terfering substance  so  far  reported. 

11.  Salting-out  Experiments. —  (a)  To  25  c.c.  of  egg  albumin 
solution  in  a  small  beaker  add  solid  ammonium  sulphate  to  the  point 
of  saturation,  keeping  the  temperature  of  the  solution  below  400  C. 
Filter,  test  the  precipitate  by  Millon's  reaction  and  the  filtrate  by  the 
biuret  test.  What  are  your  conclusions?  (b)  Repeat  the  above  ex- 
periment making  the  saturation  with  solid  sodium  chloride.  How 
does  this  result  differ  from  the  result  of  the  saturation  with  am- 
monium sulphate?  Add  2-3  drops  of  acetic  acid.  What  occurs? 
All  proteins  except  peptones  are  precipitated  by  saturating  their  so- 
lutions with  ammonium  sulphate.  Globulins  are  the  only  proteins 
precipitated  by  saturating  with  sodium  chloride  (see  Globulins,  page 
102),  unless  the  saturated  solution  is  subsequently  acidified,  in  which 
event  all  proteins  except  peptones  are  precipitated. 

Soaps  may  be  salted-out  in  a  similar  manner  (see  p.  137). 

12.  Coagulation  or  Boiling  Test. — Heat  25  c.c.  of  dilute  egg 
albumin  solution  to  the  boiling-point  in  a  small  evaporating  dish. 
The  albumin  coagulates.  Complete  coagulation  may  be  obtained  by 
acidifying  the  solution  with  3-5  drops  of  acetic  acid1  at  the  boiling- 
point.  Test  the  coagulum  by  Millon's  reaction.  The  acid  is  added 
to  neutralize  any  possible  alkalinity  of  the  solution,  and  to  dissolve 
any  substances  which  are  not  albumin  (see  further  discussion  on 
page  316). 

1  Nitric  acid  is  often  used  in  place  of  acetic  acid  in  this  test.  In  case  nitric 
acid  is  used,  ordinarily  1-2  drops  is  sufficient. 


IOO 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  32. 


13.  Coagulation  Temperature. — Prepare  4  test-tubes  each  con- 
taining 5  c.c.  of  neutral  egg  albumin  solution.  To  the  first  add  1 
drop  of  0.2  per  cent  hydrochloric  acid,  to  the  second  add  1  drop  of 

0.5  per  cent  sodium  carbonate  solution, 
to  the  third  add  1  drop  of  10  per.  cent 
sodium  chloride  solution  and  leave 
the  fourth  neutral  in  reaction.  Partly 
fill  a  beaker  of  medium  size  with 
water  and  place  it  within  a  second 
larger  beaker  which  also  contains 
water,  the  two  vessels  being  separated 
by  pieces  of  cork.  Fasten  the  four 
test-tubes  compactly  together  by  means 
of  a  rubber  band,  lower  them  into  the 
water  of  the  inner  beaker  and  suspend 
them,  by  means  of  a  clamp  attached  to 
one  of  the  tubes,  in  such  a  manner 
that  the  albumin  solutions  shall  be 
midway  between  the  upper  and  lower 
surfaces  of  the  water.  In  one  of  the 
tubes  place  a  thermometer  with  its  bulb 
entirely  beneath  the  surface  of  the 
albumin  solution  (Fig.  32).  Gently 
heat  the  water  in  the  beakers,  noting 
carefully  any  changes  which  may 
occur  in  the  albumin  solutions  and 
record  the  exact  temperature  at  which 
these  changes  occur.  The  first  ap- 
pearance of  an  opacity  in  an  albumin 
solution  indicates  the  commencement 
of  coagulation  and  the  temperature  at  which  this  occurs  should  be 
recorded  as  the  coagulation  temperature  for  that  particular  albu- 
min solution. 

What  is  the  order  in  which  the  four  solutions  coagulate? 
Repeat  the  experiment,  adding  to  the  first  tube  1  drop  of  acetic 
acid,  to  the  second   1   drop  of  concentrated  potassium  hydroxide 
solution,  to  the  third  2  drops  of  a  10  per  cent  sodium  chloride  solu- 
tion and  leave  the  fourth  neutral  as  before. 

What  is  the. order  of  coagulation  here?    Why? 
14.  Precipitation  by  Alcohol. — Prepare  3  test-tubes  each  con- 
taining about  10  c.c.  of  95  per  cent  alcohol.     To  the  first  add  one 


Coagulation    Temperature 
Apparatus. 


PROTEINS.  IOI 

drop  of  0.2  per  cent  hydrochloric  acid,  to  the  second  one  drop 
of  potassium  hydroxide  solution  and  leave  the  third  neutral  in  reac- 
tion. Add  to  each  tube  a  few  drops  of  egg  albumin  solution  and 
note  the  results.  What  do  you  conclude  from  this  experiment? 
Alcohol  precipitates  proteins  unaltered  but  if  allowed  to  remain 
under  alcohol  the  protein  is  transformed.  The  "fixing"  of  tissues 
for  histological  examination  by  means  of  alcohol  is  an  illustra- 
tion of  the  application  of  this  transformation  produced  by  alcohol. 
It  apparently  is  a  process  of  dehydrolysis. 

15.  Preparation  of  Powdered  Egg  Albumin. — This  may  be 
prepared  as  follows :  Ordinary  egg-white  finely  divided  by  means 
of  scissors  or  a  beater  is  treated  with  four  volumes  of  water  and 
filtered.  The  filtrate  is  evaporated  on  a  water-bath  at  about  500  C. 
and  the  residue  powdered  in  a  mortar. 

16.  Tests  on  Powdered  Egg  Albumin. — With  powdered  albu- 
min prepared  as  described  above  (by  yourself  or  furnished  by  the 
instructor),  try  the  following  tests: 

(a)  Solubility. 

(b)  Millon's  Reaction. 

(c)  Hopkins-Cole  Reaction. — When  used  to  detect  the  presence 
of  protein  in  solid  form  this  reaction  should  be  conducted  as  follows : 
Place  5  c.c.  of  concentrated  sulphuric  acid  in  a  test-tube  and  add 
carefully,  by  means  of  a  pipette,  3-5  c.c.  of  Hopkins-Cole  reagent. 
Introduce  a  small  amount  of  the  solid  substance  to  be  tested, 
agitate  the  tube  slightly,  and  note  that  the  suspended  pieces  assume 
a  reddish-violet  color,  which  is  the  characteristic  end-reaction  of 
the  Hopkins-Cole  test;  later  the  solution  will  also  assume  the 
reddish-violet  color. 

(d)  Composition  Test. — Heat  some  of  the  powder  in  a  test- 
tube  in  which  is  suspended  a  strip  of  moistened  red  litmus  paper 
and  across  the  mouth  of  which  is  placed  a  piece  of  filter  paper 
moistened  with  plumbic  acetic  solution.  As  the  powder  is  heated 
it  chars,  indicating  the  presence  of  carbon;  the  fumes  of  ammonia 
are  evolved,  turning  the  red  litmus  paper  blue  and  indicating  the 
presence  of  nitrogen  and  hydrogen;  the  plumbic  acetate  paper  is 
blackened,  indicating  the  presence  of  sulphur,  and  the  deposition 
of  moisture  on  the  side  of  the  tube  indicates  the  presence  of 
hydrogen. 

(e)  Immerse  a  dry  test-tube  containing  a  little  powdered  egg 
albumin  in  boiling  water  for  a  few  moments.  Remove  and  test 
the  solubility  of  the  albumin  according  to  the  directions  given  under 


102  PHYSIOLOGICAL    CHEMISTRY. 

(a)  p.  ioi.  It  is  still  soluble.  Why  has  it  not  been  coagulated? 
Repeat  the  above  experiments  with  powdered  serum  albumin  and 
see  how  the  results  compare  with  those  just  obtained. 

SULPHUR   IN    PROTEIN. 

Sulphur  is  believed  to  be  present  in  two  different  forms  in  the 
protein  molecule.  The  first  form,  which  is  present  in  greatest 
amount,  is  that  loosely  combined  with  carbon  and  hydrogen.  Sul- 
phur in  this  form  is  variously  termed  unoxidized,  loosely  combined, 
mercaptan  and  lead-blackening  sulphur.  The  second  form  is  com- 
bined in  a  more  stable  manner  with  carbon  and  oxygen  and  is 
known  as  oxidised  or  acid  sulphur.  The  protamines  are  the  only 
class  of  sulphur-free  proteins. 

Tests  for  Sulphur. 

i.  Test  for  Loosely  Combined  Sulphur. — To  equal  volumes 
of  KOH  and  egg  albumin  solutions  in  a  test-tube  add  1-2  drops  of 
plumbic  acetate  solution  and  boil  the  mixture.  Loosely  combined 
sulphur  is  indicated  by  a  darkening  of  the  solution,  the  color  deep- 
ening into  a  black  if  sufficient  sulphur  is  present.  Add  hydrochloric 
acid  and  note  the  characteristic  odor  evolved  from  the  solution. 
Write  the  reactions  for  this  test. 

2.  Test  for  Total  Sulphur  (Loosely  Combined  and  Oxidized). 
— Place  the  substance  to  be  examined  (powdered  egg  albumin) 
in  a  small  porcelain  crucible,  add  a  suitable  amount  of  solid  fusion 
mixture  (potassium  hydroxide  and  potassium  nitrate  mixed  in  the 
proportion  5:1)  and  heat  carefully  until  a  colorless  mixture  results. 
(Sodium  peroxide  may  be  used  in  place  of  this  fusion  mixture  if 
desired.)  Cool,  dissolve  the  cake  in  a  little  warm  water  and  filter. 
Acidify  the  filtrate  with  hydrochloric  acid,  heat  it  to  the  boiling- 
point  and  add  a  small  amount  of  barium  chloride  solution.  A 
white  precipitate  forms  if  sulphur  is  present.  What  is  this  pre- 
cipitate? 

GLOBULINS. 

Globulins  are  simple  proteins  especially  predominant  in  the  vege- 
table kingdom.  They  are  closely  related  to  the  albumins  and  in 
common  with  them  give  all  the  ordinary  protein  tests.  Globulins 
differ  from  the  albumins  in  being  insoluble  in  pure  (salt-free)  water. 
They  are,  however,  soluble  in  neutral  solutions  of  salts  of  strong 
bases  with  strong  acids.    Most  globulins  are  precipitated  from  their 


PROTEINS.  I03» 

solutions  by  saturation  with  solid  sodium  chloride  or  magnesium 
sulphate.  As  a  class  they  are  much  less  stable  than  the  albumins, 
a  fact  shown  by  the  increasing  difficulty  with  which  a  globulin 
dissolves  during  the  course  of  successive  reprecipitations. 

We  have  used  an  albumin  of  animal  origin  (egg  albumin)  for 
all  the  protein  tests  thus  far,  whereas  the  globulin  to  be  studied 
will  be  prepared  from  a  vegetable  source.  There  being  no  essential 
difference  between  animal  and  vegetable  proteins,  the  vegetable 
globulin  we  shall  study  may  be  taken  as  a  true  type  of  all  globulins, 
both  animal  and  vegetable. 

Experiments  on  Globulin. 

Preparation  of  the  Globulin. — Extract  20-30  grams  fa  hand- 
ful) of  crushed  hemp  seed  with  a  5  per  cent  solution  of  sodium  chlo- 
ride for  one-half  hour  at  60 °  C.  Filter  while  hot  through  a  paper 
moistened  with  5  per  cent  sodium  chloride  solution.  Place  the 
nitrate  in  the  water-bath  at  60 °  C.  and  allow  it  to  stand  for  24 
hours  in  order  that  the  globulin  may  crystallize  slowly.  In  case 
the  filtrate  is  cloudy  is  should  be  warmed  to  6o°  C.  in  order  to 
produce  a  clear  solution.  The  globulin  is  soluble  in  hot  5  per  cent 
sodium  chloride  solution  and  is  thus  extracted  from  the  hemp  seed, 
but  upon  cooling  this  solution  much  of  the  globulin  separates  in 
crystalline  form.  This  particular  globulin  is  called  edestin.  It 
crystallizes  in  several  different  forms,  chiefly  octahedra  (see  Fig. 
33,  page  104).  (The  crystalline  form  of  excelsin,  a  protein  ob- 
tained from  the  Brazil  nut,  is  shown  in  Fig.  34,  page  105.  This 
vegetable  protein  crystallizes  in  the  form  of  hexagonal  plates.) 
Filter  off  the  edestin  and  make  the  following  tests  on  the  crystalline 
body  and  on  the  filtrate  which  still  contains  some  of  the  extracted 
globulin. 

Tests  on  Crystallized  Edestin. —  (1)  Microscopical  examina- 
tion (see  Fig.  33,  p.  104). 

(2)  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see 
page  23).  Keep  these  solubilities  in  mind  for  comparison  with 
those  of  edestan,  to  be  made  later  (see  page  109). 

(3)  Milloris  Reaction. 

(4)  Coagulation  Test. — Place  a  small  amount  of  the  globulin  in 
a  test-tube,  add  a  little  water  and  boil.  Now  add  dilute  hydrochloric 
acid  and  note  that  the  protein  no  longer  dissolves.  It  has  been 
coagulated. 


#  IO4  PHYSIOLOGICAL    CHEMISTRY. 

(5)  Dissolve  the  remainder  of  the  edestin  in  0.2  per  cent  hydro- 
chloric acid  and  preserve  this  acid  solution  for  use  in  the  experiments 
on  proteans  (see  page  109). 

Fig.  33. 


Edestin. 

Tests  on  Edestin  Filtrate. —  (1)  Influence  of  Protein  Pre- 
cipitants. — Try  a  few  protein  precipitants  such  as  nitric  acid,  tannic 
acid,  picric  acid  and  mercuric  chloride. 

(2)  Biuret  Test. 

(3)  Coagulation  Test. — Boil  some  of  the  filtrate  in  a  test-tube. 
What  happens  ? 

(4)  Saturation  with  Sodium  Chloride. — Saturate  some  of  the 
filtrate  with  solid  sodium  chloride.  How  does  this  result  differ 
from  that  obtained  upon  saturating  egg  albumin  solution  with  solid 
sodium  chloride? 

(5)  Precipitation  by  Dilution. — Dilute  some  of  the  filtrate  with 
10-15  volumes  of  water.     Why  does  the  globulin  precipitate? 

Glutelins. 

It  has  been  repeatedly  shown,  particularly  by  Osborne,  that  after 
extracting  the  seeds  of  cereals  with  water,  neutral  salt  solution  and 
strong  alcohol,  there  still  remains  a  residue  which  contains  protein 
material  which  may  be  extracted  by  very  dilute  acid  or  alkali. 
These  proteins  which  are  insoluble  in  all  neutral  solvents,  but  readily 
soluble  in  very  dilute  acids  and  alkalis  are  called  glutelins.  The 
only  member  of  the  group  which  has  yet  received  a  name,  is  the 
glutenin  of  wheat,  a  protein  which  constitutes  nearly  fifty  per  cent 


PROTEINS. 


I05 


of  the  gluten.     It  is  not  definitely  known  whether  glutelins  occur 
as  constituents  of  all  seeds. 

Prolamins  (Alcohol-soluble  Proteins). 

The  term  prolamin  has  been  proposed  by  Osborne,  for  the  group 
of  proteins  formerly  termed  "alcohol-soluble  proteins."  The  name 
is  very  appropriate  inasmuch  as  these  proteins  yield,  upon  hydroly- 
sis, especially  large  amounts  of  proline  and  ammonia.  The  pro- 
lamins are  simple  proteins  which  are  insoluble  in  water,  absolute 
alcohol  and  other  neutral  solvents,  but  are  soluble  in  70  to  80  per 
cent  alcohol  and  in  dilute  acids  and  alkalis.  They  occur  widely  dis- 
tributed, particularly  in  the  vegetable  kingdom.  The  only  prolamins 
yet  described  are  the  zein  of  maize,  the  hordein  of  barley,  the  gliadin 
of  wheat  and  rye  and  the  bynin  of  malt.  They  yield  relatively  large 
amounts  of  glutamic  acid  on  hydrolysis  but  no  lysin.     The  largest 


Fig.  34. 


EXCELSIN,  THE  PROTEIN   OF  THE  BRAZIL  NUT. 

(Drawn  from  crystals  furnished  by  Dr.  Thomas  B.  Osborne,  New  Haven,  Conn.) 

percentage  of  glutamic  acid  (41.32  per  cent)  ever  obtained  as  a 
decomposition  product  of  a  protein  substance  has  very  recently  been 
obtained  by  Kleinschmitt  from  the  hydrolysis  of  the  prolamin 
hordein.1  This  yield  of  glutamic  acid  is  also  the  largest  amount 
of  any  single  decomposition  product  yet  obtained  from  any  protein 
except  protamines. 

'Up  to  this  time  the  yield  of  37.33  per  cent  obtained  by  Osborne  and  Harris 
from  gliadin,  was  the  maximum  yield. 


106  PHYSIOLOGICAL    CHEMISTRY. 

Albuminoids.     (Scleroproteins.) 

The  albuminoids  yield  similar  hydrolytic  products  to  those  ob- 
tained from  the  other  simple  proteins  already  considered,  thus  indi- 
cating that  they  possess  essentially  the  same  chemical  structure. 
They  differ  from  all  other  proteins,  whether  simple,  conjugated  or 
derived,  in  that  they  are  insoluble  in  all  neutral  solvents.  The 
albuminoids  include  "the  principal  organic  constituents  of  the 
skeletal  structure  of  animals  as  well  as  their  external  covering  and 
its  appendages."  Some  of  the  principal  albuminoids  are  keratin, 
elastin,  collagen,  reticulin,  spongin,  and  fibroin.  Gelatin  cannot 
be  classed  as  an  albuminoid  although  it  is  a  transformation  product 
of  collagen.  The  various  albuminoids  differ  from  each  other  in 
certain  fundamental  characteristics  which  will  be  considered  in 
detail  under  Epithelial  and  Connective  Tissue  (see  Chapter  XIV, 
p.  227). 

CONJUGATED   PROTEINS. 

Conjugated  proteins  consist  of  a  protein  molecule  united  to  some 
other  molecule  or  molecules  otherwise  than  as  a  salt.  We  have 
glycoproteins,  nude  0  proteins,  hcemoglobins  (chromoproteins), 
phosphoproteins  and  lecitho proteins  as  the  five  classes  of  conju- 
gated proteins. 

Glycoproteins  may  be  considered  as  compounds  of  the  protein 
molecule  with  a  substance  or  substances  containing  a  carbohydrate 
group  other  than  a  nucleic  acid.  The  glycoproteins  yield,  upon 
decomposition,  protein  and  carbohydrate  derivatives,  notably  gly- 
cosamine,  CH2OH- (CHOH)3-CH(NH2) -CHO,  and  galactosa- 
mine,  OHCH2- (CHOH)3-CH(NH2) -CHO.  The  principal  gly- 
coproteins are  mucoids,  mucins  and  chondro proteins.  By  the  term 
mucoid  we  may  designate  those  glycoproteins  which  occur  in  tis- 
sues, such  as  tendomucoid  from  tendinous  tissue  and  osseomucoid 
from  bone.  The  elementary  composition  of  these  typical  mucoids 
is  as  follows : 

N.  S.  C.  H.  O. 

Tendomucoid  11.75      2.33      48.76      6.53      30.60     (Chittenden  and  Gies) 

Osseomucoid    12.22      2.32      47.43      6.63      31.40 

The  term  mucins  may  be  said  to  include  those  forms  of  glyco- 
proteins which  occur  in  the  secretions  and  fluids  of  the  body. 
Chondroproteins  are  so  named  because  chondromucoid,  the  prin- 
cipal member  of  the  group,  is  derived  from  cartilage  (chondrigen). 
Amyloid,  which  appears  pathologically  in  the  spleen,  liver  and 
kidneys,  is  also  a  chondroprotein. 


PROTEINS.  I07 

The  nucleoproteins  occur  principally  in  animal  and  vegetable 
cells,  and  following  the  destruction  of  these  cells  they  are  Eound 
in  the  fluids  of  the  body.  These  proteins  are  discharged  into  the 
tissue  fluids  by  the  activity  or  disintegration  of  cells.  Combined 
with  the  simple  protein  in  the  nucleoprotein  molecule  we  find 
nucleic  acid,  a  body  which  contains  phosphorus  and  which  yields 
purine  bases  and  pyrimidine  bases  (thymine,  cytosine  and  uracil) 
upon  decomposition.  The  so-called  nucleins  are  formed  in  the  gas- 
tric digestion  of  nucleoproteins. 

Wheeler-Johnson  Reaction  for  Uracil  and  Cytosine. — To 
about  5  c.c.  of  the  solution  under  examination  add  bromine  water 
until  the  color  is  permanent.1  In  case  the  solution  contains  only 
small  quantities  of  cytosine  or  uracil,  it  is  advisable  to  remove  the 
excess  of  bromine  by  passing  a  stream  of  air  through  the  solution. 
Now  add  an  excess  of  an  aqueous  solution  of  barium  hydroxide 
and  note  the  appearance  of  a  purple  color. 

Very  dilute  solutions  do  not  give  the  test.  Under  these  condi- 
tions the  solution  should  be  evaporated  to  dryness,  the  residue  dis- 
solved in  a  little  bromine  water  and  the  excess  of  bromine  removed. 
Then  upon  adding  an  excess  of  barium  hydroxide  a  decided  bluish- 
pink  or  lavender  color  will  appear  in  the  presence  of  as  small  an 
amount  as  0.00 1  gram  of  uracil. 

In  testing  solutions  for  cytosine,  it  is  preferable  to  warm  or  boil 
the  solution  with  bromine  water,  and  after  cooling  the  solution  to 
apply  the  test  as  suggested  above,  being  careful  to  have  a  slight 
excess  of  bromine  present  before  adding  barium  hydroxide. 

The  phospho proteins  are  called  nude 0 albumins  in  many  classi- 
fications and  are  grouped  among  the  simple  proteins.  They  are 
considered  to  be  "  compounds  of  the  protein  molecule  with  some, 
as  yet  undefined,  phosphorus-containing  substances  other  than  a 
nucleic  acid  or  lecithin."  The  percentage  of  phosphorus  in  phos- 
phoproteins  is  very  similar  to  that  in  nucleoproteins  but  they  differ 
from  this  latter  class  of  proteins  in  that  they  do  not  yield  any 
purine  bases  upon  hydrolytic  cleavage.  Two  of  the  common  phos- 
phoproteins  are  the  caseinogen  of  milk  and  the  ovovitellin  of  the 
egg-yolk. 

The  hemoglobins  (chromoproteins)  are  compounds  of  the  pro- 
tein molecule  with  hsematin  or  some  similar  substance.     The  prin- 

1  Avoid  the  addition  of  a  large  excess  of  bromine  inasmuch  as  this  will 
interfere  with  the  test. 


108  PHYSIOLOGICAL    CHEMISTRY. 

cipal  member  of  the  group  is  the  haemoglobin  of  the  blood.  Upon 
hydrolytic  cleavage  this  haemoglobin  yields  a  protein  termed  globin 
and  a  coloring  matter  termed  hcemochromogen.  The  latter  sub- 
stance contains  iron  and  upon  coming  in  contact  with  oxygen  is 
oxidized  to  form  hcematin.  Hcemocyanin,  another  member  of  the 
class  of  haemoglobins,  occurs  in  the  blood  of  certain  invertebrates, 
notably  cephalopods,  gasteropods,  and  Crustacea.  Haemocyanin 
generally  contains  either  copper,  manganese,  or  zinc  in  place  of  the 
iron  of  the  haemoglobin  molecule. 

The  lecitho proteins  include  such  substances  as  lecithans  and 
phosphophatides  which  consist  of  a  protein  molecule  joined  to  leci- 
thin. They  have  been  comparatively  little  studied  until  recently, 
and  in  much  of  the  older  research  they  were  undoubtedly  con- 
sidered as  lecithins. 

For  experiments  on  conjugated  proteins  see  pages  56,  195,  196, 
199,  201,  203,  223  and  228. 

DERIVED    PROTEINS. 

These  substances  are  derivatives  which  are  formed  through  hy- 
drolytic changes  of  the  original  protein  molecule.  They  may  be 
divided  into  two  groups,  the  primary  protein  derivatives  and  the 
secondary  protein  derivatives.  The  term  secondary  derivatives  is 
made  use  of  in  this  connection  since  the  formation  of  the  primary 
derivatives  generally  precedes  the  formation  of  these  secondary 
derivatives.  These  derived  proteins  are  obtained  from  native 
simple  proteins  by  hydrolyses  of  various  kinds,  e.  g.,  through  the 
action  of  acids,  alkalis,  heat  or  enzymes.  The  particular  class  of 
derived  protein  desired  regulates  the  method  of  treatment  to  which 
the  native  protein  is  subjected. 

Primary  Protein  Derivatives. 

The  primary  protein  derivatives  are  "  apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alterations  of  the 
protein  molecule."  This  class  includes  proteans,  metaproteins,  and 
coagulated  proteins. 

PROTEANS. 

Proteans  are  those  insoluble  protein  substances  which  are  pro- 
duced from  proteins  originally  soluble  through  the  incipient  action 
of  water,  enzymes,  or  very  dilute  acids.     It  is  well  known  that 


PROTEINS.  IO9 

globulins  become  insoluble  upon  repeated  reprecipitation  and  it  may 
possibly  be  found  that  the  greater  number  of  the  proteans  are 
transformed  globulins.  Osborne,  however,  believes  that  nearly  all 
proteins  may  give  rise  to  proteans.  This  investigator  who  has  so 
very  thoroughly  investigated  many  of  the  vegetable  proteins  claims 
that  the  hydrogen  ion  is  the  active  agent  in  the  transformation. 
The  protein  produced  from  the  transformation  of  edestin  is  called 
edestan,  that  produced  from  myosin  is  called  myosan,  etc.  The 
name  protean  was  first  given  to  this  class  of  proteins  by  Osborne 
in  1900  in  connection  with  his  studies  of  edestin. 

Experiments  on  Proteans. 
Preparation  and  Study  of  Edestan. — Prepare  edestin  accord- 
ing to  the  directions  given  on  page  103.  Bring  the  edestin  into  solu- 
tion in  0.2  per  cent  hydrochloric  acid  and  permit  the  acid  solution 
to  stand  for  about  one-half  hour.1  Neutralize,  with  a  0.5  per  cent 
solution  of  sodium  carbonate,  filter  off  the  precipitate  of  edestan 
and  make  the  following  tests : 

1.  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see 
page  23).  Note  the  altered  solubility  of  the  edestan  as  compared 
with  that  of  edestin  (see  page  103). 

2.  Millon's  Reaction. 

3.  Coagulation  Test. — Place  a  small  amount  of  the  protean  in 
a  test-tube,  add  a  little  water  and  boil.  Now  add  dilute  hydro- 
chloric acid  and  note  that  the  protein  no  longer  dissolves.  It  has 
been  coagulated. 

4.  Tests  on  Edestan  Solution. — Dissolve  the  remainder  of  the 
edestan  precipitate  in  0.2  per  cent  hydrochloric  acid  and  make  the 
following  tests : 

(a)  Biuret  Test. 

(b)  Influence  of  Protein  Precipitants. — Try  a   few   protein 
precipitants  such  as  picric  acid  and  mercuric  chloride. 

METAPROTEINS. 

The  metaproteins  are  formed  from  the  native  simple  proteins 
through  an  action  similar  to  that  by  which  proteans  are  formed. 
In  the  case  of  the  metaproteins,  however,  the  changes  in  the  origi- 
nal protein  molecule  are  more  profound.  These  derived  proteins 
are  characterized  by  being  soluble  in  very  weak  acids  and  alkalis, 

lThe  edestan  solution  preserved  from  experiment  (5),  page  104,  may  be  used. 


IIO  PHYSIOLOGICAL    CHEMISTRY. 

but  insoluble  in  neutral  fluids.  The  metaproteins  have  generally 
been  termed  albuminates,  but  inasmuch  as  the  termination  ate  sig- 
nifies a  salt  it  has  always  been  somewhat  of  a  misnomer. 

Two  of  the  principal  metaproteins  are  the  acid  metaprotein  or 
so-called  acid  albuminate  and  the  alkali  metaprotein  or  so-called 
alkali  albuminate.  They  differ  from  the  native  simple  proteins 
principally  in  being  insoluble  in  sodium  chloride  solution  and  in  not 
being  coagulated  except  when  suspended  in  neutral  fluids.  Both  forms 
of  metaprotein  are  precipitated  upon  the  approximate  neutraliza- 
tion of  their  solutions.  They  are  precipitated  by  saturating  their 
solutions  with  ammonium  sulphate,  and  by  sodium  chloride,  also, 
provided  they  are  dissolved  in  an  acid  solution.  Acid  metaprotein 
contains  a  higher  percentage  of  nitrogen  and  sulphur  than  the 
alkali  metaprotein  from  the  same  source,  since  some  of  the  nitro- 
gen and  sulphur  of  the  original  protein  is  liberated  in  the  forma- 
tion of  the  latter.  Because  of  this  fact,  it  is  impossible  to  trans- 
form an  alkali  metaprotein  into  an  acid  metaprotein,  while  it  is 
possible  to  reverse  the  process  and  transform  the  acid  metaprotein 
into  the  alkali  modification. 

Experiments    on    Metaproteins. 

ACID  METAPROTEIN  (ACID  ALBUMINATE). 

Preparation  and  Study. — Take  25  grams  of  hashed  lean  beef, 
washed  free  from  the  major  portion  of  blood  and  inorganic  matter, 
and  place  it  in  a  medium-sized  beaker  with  100  c.c.  of  0.2  per  cent 
HC1.  Place  it  on  a  boiling  water-bath  for  one-half  hour,  filter, 
cool  and  divide  the  filtrate  into  two  parts.  Neutralize  the  first  part 
with  dilute  KOH  solution,  filter  off  the  precipitate  of  acid  meta- 
protein and  make  the  following  tests  : 

( 1 )  Solubility. — Solubility  in  the  ordinary  solvents  (see  page  23  ) . 

(2)  Milton's  Reaction. 

(3)  Coagulation  Test. — Suspend  a  little  of  the  metaprotein  in 
water  (neutral  solution)  and  heat  to  boiling  for  a  few  moments. 
Now  add  1-2  drops  of  KOH  solution  to  the  water  and  see  if  the 
metaprotein  is  still  soluble  in  dilute  alkali.  What  is  the  result 
and  why? 

(4)  Test  for  Loosely  Combined  Sulphur  (see  page  102). 

Subject  the  second  part  of  the  original  solution  to  the  following 
tests : 


PROTEINS.  '  I  ' 

(i)  Coagulation  Test. — Heat  some  of  the  solution  to  boiling 
in  a  test-tube.     Does  it  coagulate  ? 

( 2  )   Biuret  Test.  • 

(3)  Influence  of  Protein  Precipitants. — Try  a  few  protein  pre- 
cipitants  such  as  picric  acid  and  mercuric  chloride.  How  do  the 
results  obtained  compare  with  those  from  the  experiments  on  egg 
albumin?     (See  page  97.) 

ALKALI  METAPROTEIN  (ALKALI  ALBUMINATE). 

Preparation  and  Study. — Carefully  separate  the  white  from 
the  yolk  of  a  hen's  egg  and  place  the  former  in  an  evaporating  dish. 
Add  concentrated  potassium  hydroxide  solution,  drop  by  drop,  stir- 
ring continuously.  The  mass  gradually  thickens  and  finally  as- 
sumes the  consistency  of  jelly.  This  is  solid  alkali  metaprotein  or 
"  Lieberkiihn's  jelly."  Do  not  add  an  excess  of  potassium  hydrox- 
ide or  the  jelly  will  dissolve.  Cut  it  into  small  pieces,  place  a  cloth 
or  wire  gauze  over  the  dish  and  by  means  of  running  water  wash 
the  pieces  free  from  adherent  alkali.  Now  add  a  small  amount  of 
water,  which  forms  a  weak  alkaline  solution  with  the  alkali  within 
the  pieces,  and  dissolve  the  jelly  by  gentle  heat.  Cool  the  solution 
and  divide  it  into  two  parts.  Proceed  as  follows  with  the  first  part: 
Neutralize  with  dilute  hydrochloric  acid,  noting  the  odor  of  the 
liberated  hydrogen  sulphide  as  the  alkali  metaprotein  precipitates. 
Filter  off  the  precipitate  and  test  as  for  acid  metaprotein,  page  no, 
noting  particularly  the  sulphur  test.  How  does  this  test  compare 
with  that  given  by  the  acid  metaprotein?  Make  tests  on  the  second 
part  of  the  solution  the  same  as  for  acid  metaprotein,  page  no. 

Coagulated  Proteins. 

These  derived  proteins  are  produced  from  unaltered  protein 
materials  by  heat,  by  long  standing  under  alcohol,  or  by  the  con- 
tinuous movement  of  their  solutions  such  as  that  produced  by 
rapid  stirring  or  shaking.  In  particular  instances,  such  as  the 
formation  of  fibrin  from  fibrinogen  (see  page  191),  the  coagula- 
tion may  be  produced  by  enzyme  action.  Ordinary  soluble  proteins 
after  having  been  transformed  into  the  coagulated  modification 
are  no  longer  soluble  in  the  ordinary  solvents.  Upon  being  heated 
in  the  presence  of  strong  acids  or  alkalis,  coagulated  proteins  are 
convened  into  metaproteins. 

'-•'-I     protems  consulate  at  an  approximately  fixed  temperature 


112  PHYSIOLOGICAL    CHEMISTRY. 

under  definite  conditions  (see  pp.  ioo  and  242).  This  characteristic 
may  be  applied  to  separate  different  coagulable  proteins  from  the  same 
solution  by  fractional  coagulation.  The  coagulation  temperature 
frequently  may  serve  in  a  measure  to  identify  proteins  in  a  manner 
similar  to  the  melting-point  or  boiling-point  of  many  other  organic 
substances.  The  separation  of  proteins  by  fractional  coagulation 
is  thus  analogous  to  the  separation  of  volatile  substances  by  means 
of  fractional  distillation.  This  method  of  separating  proteins  is 
not  a  satisfactory  one,  however,  inasmuch  as  proteins  in  solution 
have  different  effects  ,upon  one  another  and  also  because  of  the 
fact  that  the  nature  of  the  solvent  causes  a  variation  in  the  tem- 
perature at  which  a  given  protein  coagulates.  The  nature  of  the 
process  involved  in  the  coagulation  of  proteins  by  heat  is  not 
well  understood,  but  it  is  probable  that  in  addition  to  the  altered 
arrangement  of  the  component  atoms  in  the  molecule,  there  is  a 
mild  hydrolysis  which  is  accompanied  by  the  liberation  of  minute 
amounts  of  hydrogen,  nitrogen  and  sulphur.  The  presence  of  a 
neutral  salt  or  a  trace  of  a  mineral  acid  may  facilitate  the  coagula- 
tion of  a  protein  solution  (see  page  100),  whereas  any  appreciable 
amount  of  acid  or  alkali  will  retard  or  entirely  prevent  such  coagu- 
lation. 

Experiments  on  Coagulated  Protein. 

Ordinary  coagulated  egg-white  may  be  used  in  the  following 
tests : 

1.  Solubility. — Try  the  solubility  of  small  pieces  of  the  coagu- 
lated protein  in  each  of  the  ordinary  solvents  (see  page  23). 

2.  Millon's  Reaction. 

3.  Xanthoproteic  Reaction. — Partly  dissolve  a  medium-sized 
piece  of  the  protein  in  concentrated  nitric  acid.  Cool  the  solution 
and  add  an  excess  of  ammonium  hydroxide.  Both  the  protein 
solution  and  the  undissolved  protein  will  be  colored  orange. 

4.  Biuret  Test. — Partly  dissolve  a  medium-sized  piece  of  the 
protein  in  concentrated  potassium  hydroxide  solution.  If  the  proper 
dilution  of  cupric  sulphate  solution  is  now  added  the  white  coagu- 
lated protein,  as  well  as  the  protein  solution,  will  assume  the  char- 
acteristic purplish-violet  color. 

5.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  10 1. 


PROTEINS.  I  I  3 

Secondary  Protein  Derivatives. 

These  derivatives  result  from  a  more  profound  cleavage  of  the 
protein  molecule  than  that  which  occurs  in  the  formation  of  the 
primary  derivatives.  The  class  includes  proteoses,  peptones  and 
peptides. 

PROTEOSES  AND  PEPTONES. 

Proteoses  are  intermediate  products  in  the  digestion  of  proteins 
by  proteolytic  enzymes,  as  well  as  in  the  decomposition  of  proteins 
by  hydrolysis  and  the  putrefaction  of  proteins  through  the  action 
of  bacteria.  Proteoses  are  also  called  albumoses  by  some  writers, 
but  it  seems  more  logical  to  reserve  the  term  albumose  for  the 
proteose  of  albumin. 

Peptones  are  formed  after  the  proteoses  and  it  has  been  cus- 
tomary to  consider  them  as  the  last  product  of  the  processes  before 
mentioned  which  still  possess  true  protein  characteristics.  In  other 
words  it  has  been  considered  that  the  protein  nature  of  the  end- 
products  of  the  cleavage  of  the  protein  molecule  ceased  with  the 
peptones,  and  that  the  simpler  bodies  formed  from  peptones  were 
substances  of  a  different  nature  (see  page  63).  However,  as  the 
end-products  have  been  more  carefully  studied,  it  has  been  found 
to  be  no  easy  matter  to  designate  the  exact  character  of  a  peptone 
or  to  indicate  the  exact  point  at  which  the  peptone  characteristic 
ends  and  the  peptide  characteristic  begins.  The  situation  regarding 
the  proteoses,  peptones  and  peptides,  is  at  present  a  most  unsatis- 
factory one  because  of  the  unsettled  state  of  our  knowledge  regard- 
ing them.  The  exact  differences  between  certain  members  of  the 
peptone  and  peptide  groups  remain  to  be  more  accurately  estab- 
lished. It  has  been  quite  well  established  that  the  peptones  are 
peptides  or  mixtures  of  peptides,  but  the  term  peptide  is  used  at 
present  to  designate  only  those  possessing  a  definite  structure. 

There  are  several  proteoses  (protoproteose,  heteroproteose  and 
deuteroproteose),  and  at  least  two  peptones  (amphopeptone  and 
antipeptone),  which  result  from  proteolysis.  The  differentiation 
of  the  various  proteoses  and  peptones  at  present  in  use  is  rather 
unsatisfactory.  These  compounds  are  classified  according  to  their 
varying  solubilities,  especially  in  ammonium  sulphate  solutions  of 
different  strengths.  The  exact  differences  in  composition  between 
the  various  members  of  the  group  remain  to  be  more  accurately 
established.  Because  of  the  difficulty  attending  the  separation  of 
these  bodies,  pure  proteose  and  peptone  are  not  easy  to  procure. 
9 


114  PHYSIOLOGICAL    CHEMISTRY. 

The  so-called  peptones  sold  commercially  contain  a  large  amount 
of  proteose.  As  a  class  the  proteoses  and  peptones  are  very  soluble, 
diffusible  bodies  which  are  non-coagulable  by  heat.  Peptones  differ 
from  proteoses  in  being  more  diffusible,  non-precipitable  by 
(NH4)2S04,  and  by  their  failure  to  give  any  reaction  with  potas- 
sium ferrocyanide  and  acetic  acid,  potassio-mer curie  iodide  and 
HC1,  picric  acid,  and  trichloracetic  acid.  The  so-called  primary 
proteoses  are  precipitated  by  HN03  and  are  the  only  members  of 
the  proteose-peptone  group  which  are  so  precipitated. 

Some  of  the  more  general  characteristics  of  the  proteose-peptone 
group  may  be  noted  by  making  the  following  simple  tests  on  a 
proteose-peptone  powder : 

( i )   Solubility. — Solubility  in  the  ordinary  solvents  ( see  page  23 ) . 

(2)   Millon's  Reaction. 

Dissolve  a  little  of  the  powder  in  water  and  test  the  solution  as 
follows : 

(1)  Precipitation  by  Picric  Acid. — To  5  c.c.  of  proteose-peptone 
solution  in  a  test-tube  add  picric  acid  until  a  permanent  precipi- 
tate forms.  The  precipitate  disappears  on  heating  and  returns 
on  cooling. 

(2)  Precipitation  by  a  Mineral  Acid. — Try  the  precipitation  by 
nitric  acid. 

(3)  Coagulation  Test. — Heat  a  little  proteose-peptone  solution 
to  boiling.    Does  it  coagulate  like  the  other  simple  proteins  studied  ? 

SEPARATION  OF  PROTEOSES  AND  PEPTONES.1 

Place  50  c.c.  of  proteose-peptone  solution  in  an  evaporating  dish 
or  casserole,  and  half-saturate  it  with  ammonium  sulphate  solution, 
which  may  be  accomplished  by  adding  an  equal  volume  of  saturated 
ammonium  sulphate  solution.  At  this  point  note  the  appearance 
of  a  precipitate  of  the  primary  proteoses  (protoproteose  and  hetero- 
proteose).  Now  heat  the  half-saturated  solution  and  its  suspended 
precipitate  to  boiling  and  saturate  the  solution  with  solid  ammo- 
nium sulphate.  At  full  saturation  the  secondary  proteoses  (deu- 
teroproteoses)  are  precipitated.     The  peptones  remain  in  solution. 

Proceed  as  follows  with  the  precipitate  of  proteoses :  Collect 
the  sticky  precipitate  on  a  rubber-tipped  stirring  rod  or  remove  it 
by  means  of  a  watch  glass  to  a  small  evaporating  dish  and  dissolve 

1  The  separation  of  proteoses  and  peptones  by  means  of  fractional  precipitation 
with  ammonium  sulphate  does  not  possess  the  significance  it  once  possessed 
inasmuch  as  the  boundary  between  these  substances  and  peptides  is  not  well 
defined  (see  p.  113). 


PROTEINS.  I  I  5 

it  in  a  little  water.  To  remove  the  ammonium  sulphate,  which 
adhered  to  the  precipitate  and  is  now  in  solution,  add  barium  car- 
bonate, boil,  and  filter  off  the  precipitate  of  barium  sulphate.  Con- 
centrate the  proteose  solution  to  a  small  volume1  and  make  the 
following  tests : 
(i)  Biuret  Test. 

(2)  Precipitation  by  Nitric  Acid. — What  would  a  precipitate  at 
this  point  indicate? 

(3)  Precipitation  by  Trichloracetic  Acid. — This  precipitate  dis- 
solves on  heating  and  returns  on  cooling. 

(4)  Precipitation  by  Picric  Acid. — This  precipitate  also  disap- 
pears on  heating  and  returns  on  cooling. 

(5)  Precipitation  by  Potassio-mercuric  Iodide  and  Hydrochloric 
Acid. 

(6)  Coagulation  Test. — Boil  a  little  in  a  test-tube.  Does  it 
coagulate? 

(7)  Acetic  Acid  and  Potassium  Ferrocyanide  Test. 

The  solution  containing  the  peptones  should  be  cooled  and  fil- 
tered, and  the  ammonium  sulphate  in  solution  removed  by  boiling 
with  barium  carbonate  as  described  above.  After  filtering  off 
the  barium  sulphate  precipitate,  concentrate  the  peptone  filtrate  to 
a  small  volume1  and  repeat  the  test  as  given  under  the  proteose  solu- 
tion, above.  In  the  biuret  test  the  solution  should  be  made  very 
strongly  alkaline  with  solid  potassium  hydroxide. 

PEPTIDES. 

The  peptides  are  "  definitely  characterized  combinations  of  two 
or  more  amino  acids,  the  carboxyl  (COOH)  group  of  one  being 
united  with  the  amino  (NH2)  group  of  the  other  with  the  elimina- 
tion of  a  molecule  of- water."  These  peptides  are  more  fully  dis- 
cussed on  pages  66  and  113. 

REVIEW    OF   PROTEINS. 

In  order  to  facilitate  the  student's  review  of  the  proteins,  the 
preparation  of  a  chart  similar  to  the  model  on  p.  117  is  recom- 
mended. The  signs  +  and  —  may  be  conveniently  used  to  indi- 
cate positive  and  negative  reactions. 

1  If  the  proteoses  are  desired  in  powder  form,  this  concentrated  proteose  solu- 
tion may  now  be  precipitated  by  alcohol,  and  this  precipitate,  after  being  washed 
with  absolute  alcohol  and  with  ether,  may  be  dried  and  powdered. 


n6 


PHYSIOLOGICAL    CHEMISTRY. 


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117 


MODEL  CHART  FOR  REVIEW  PURPOSES. 


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2  .a 

Potassio- mercuric 
Iodide  +  HC1. 

Picric  Acid. 

Trichloracetic 

Acid. 

* 

O 
CO 

X 
Z 

0 

n 
2 

Albumin. 

Globulin. 

1 

Protean. 

j 

Acid  metaprotein. 

Alkali  metaprotein. 

Proteose. 

— 





— 









Peptone. 
Coagulated  protein. 



1 

"  Unknown  "  Mixtures  and  Solutions  of  Proteins. 

At  this  point  the  student's  knowledge  of  the  characteristics  of 
the  various  proteins  studied  will  be  tested  by  requiring  him  to 
examine  several  "unknown"  protein  mixtures  or  solutions  and 
make  full  report  upon  the  same.  The  scheme  given  on  page  116 
may  be  used  in  this  examination. 


CHAPTER    VI. 

GASTRIC    DIGESTION. 

Gastric  digestion  takes  place  in  the  stomach  and  is  promoted  by 
the  gastric  juice,  which  is  secreted  by  the  glands  of  the  stomach 
mucosa.  These  glands  are  of  two  kinds,  fundus  glands  and  pyloric 
glands  which  are  situated,  as  their  names  imply,  in  the  regions 
of  the  fundus  and  pylorus.  The  principal  foods  acted  upon  in 
gastric  digestion  are  the  proteins  which  are  so  changed  by  its  pro- 
cesses as  to  become  better  prepared  for  further  digestion  in  the 
intestine  and  for  their  final  absorption. 

From  reliable  experiments  made  upon  lower  animals  it  is  evident 
that  the  gastric  juice  is  secreted  as  the  result  of  stimuli  of  two 
forms,  i.  e.,  psychical  stimuli  and  chemical  stimuli.  The  psychical 
form  of  stimuli  may  be  produced  by  the  sight,  thought  or  taste 
of  food,  and  the  chemical  stimuli  may  be  produced  by  certain 
substances  such  as  water,  the  extractives  of  meat,  etc.,  when  com- 
ing in  contact  with  the  stomach  mucosa.  The  volume  of  gastric 
juice  secreted  during  any  given  period  of  digestion,  varies  with  the 
quantity  and  kind  of  the  food.  These  conclusions  were  deduced 
principally  from  a  series  of  so-called  delusive  feeding  experiments. 
A  dog  was  prepared  with  two  oesophageal  openings  and  a  gastric 
fistula.  When  thus  prepared  and  fed  foods  of  various  kinds  such 
as  meat  and  bread,  the  material  instead  of  passing  to  the  stomach, 
would  invariably  find  its  way  out  of  the  animal's  body  at  the  upper 
oesophageal  opening.  Through  the  medium  of  the  gastric  fistula 
the  course  of  the  secretion  of  gastric  juice  could  be  carefully 
followed.  It  was  found  that  when  the  dog  ate  meat,  for  example, 
there  was  a  large  secretion  of  gastric  juice  notwithstanding  no  por- 
tion of  the  food  eaten  had  reached  the  stomach.  Further  experi- 
ments made  through  the  medium  of  a  cul-de-sac  formed  from  the 
stomach  wall  have  given  us  many  valuable  conclusions,  among 
others  those  regarding  the  influence  of  the  chemical  stimuli.  The 
method  followed  was  to  feed  the  animal  certain  substances  and 
note  the  secretion  of  gastric  juice  in  the  miniature  stomach  while 
the  real  process  of  digestion  was  taking  place  in  the  stomach  proper. 

Normal  gastric  juice  is  a  thin,  light  colored  fluid  which  is  acid 

118 


GASTRIC    DIGESTION.  119 

in  reaction  and  has  a  specific  gravity  varying  between  i.ooi  and 
1. 010.  It  contains  only  2-3  per  cent  of  solid  matter  which  is  made 
up  principally  of  hydrochloric  acid,  sodium  chloride,  potassium 
chloride,  earthy  phosphates,  mucin  and  the  enzymes  pepsin,  gastric 
rennin  and  gastric  lipase;  the  hydrochloric  acid  and  the  enzymes 
are  of  the  greatest  importance.  The  acidity  of  the  gastric  juice  is 
due  to  free  hydrochloric  acid  which  is  secreted  by  the  parietal  cells 
of  the  fundus  as  well  as  by  the  chief  cells  of  both  the  fundus  and 
pyloric  glands,  and,  in  man,  is  generally  present  to  the  extent  of 
0.2-0.3  per  cent.  When  the  amount  of  hydrochloric  acid  varies  to 
any  considerable  degree  from  these  values  a  condition  of  hypoacidity 
or  hyperacidity  is  established.  Hydrochloric  acid  has  the  power  of 
combining  with  protein  substances  taken  in  the  food,  thus  forming 
so-called  combined  hydrochloric  acid.  This  combined  acid  is  a  less 
potent  germicide  than  free  hydrochloric  acid  and  has  less  power  to 
destroy  the  amylolytic  enzyme  salivary  amylase  (ptyalin)  of  the 
saliva.  This  last  fact  explains  to  a  degree  the  possibility  of  the 
continuance  of  salivary  digestion  in  the  stomach.  The  hydrochloric 
acid  of  the  gastric  juice  forms  a  medium  in  which  the  pepsin  can 
most  satisfactorily  digest  the  protein  food,  and  at  the  same  time 
it  acts  as  an  antiseptic  or  germicide  which  prevents  putrefactive 
processes  in  the  stomach.  It  also  possesses  the  power  of  inverting 
cane  sugar,  this  property  being  due  to  the  hydrogen  ion.  When 
the  hydrochloric  acid  of  the  gastric  juice  is  diminished  in  quan- 
tity (hypoacidity)  or  absent,  as  it  may  be  in  many  cases  of  func- 
tional or  organic  disease,  there  is  no  check  to  the  growth  of  micro- 
organisms in  the  stomach.  There  are  however  certain  of  the 
more  resistant  spores  which  even  the  normal  acidity  of  the  gastric 
juice  will  not  destroy.  A  condition  of  hypoacidity  may  also  give 
rise  to  fermentation  with  the  formation  of  comparatively  large 
amounts  of  such  substances  as  lactic  acid  and  butyric  acid. 

The  question  of  the  origin  of  the  hydrochloric  acid  of  the  gastric 
juice  is  a  problem  to  whose  solution  many  investigators  have  given 
much  attention.  Many  theories  have  been  proposed,  among  them 
being  Bunge's  mass  action  theory,  Koppe's  electrolytic  dissociation 
theory,  and  the  more  recent  theory  based  upon  the  interaction  of 
sodium  chloride  and  lactic  acid.  We  cannot  go  into  a  discussion 
of  these  various  theories.  Each  of  them  has  met  with  objection 
and  we  have,  as  yet,  no  generally  accepted  theory  as  to  the  origin 
of  the  hydrochloric  acid  of  the  gastric  juice.  That  this  hydrochloric 
acid  originates  from  the  chlorides  of  the  blood  is  apparently  a  well 


120  PHYSIOLOGICAL    CHEMISTRY. 

established  fact,  but  farther  than  this  no  positive  statement  can  be 
made. 

The  most  important  of  the  enzymes  of  the  gastric  juice  is  the 
proteolytic  enzyme  pepsin.  The  pepsin  does  not  originate  as  such 
in  the  gastric  cells  but  is  formed  from  its  precursor  the  zymo- 
gen or  mother-substance  pepsinogen  which  is  produced  by  the 
parietal  cells  of  the  fundus  as  well  as  by  the  chief  cells  of  the  fun- 
dus and  pyloric  glands.  Upon  coming  in  contact  with  the  hy- 
drochloric acid  of  the  secretion  this  pepsinogen  is  immediately 
transformed  into  pepsin.  Pepsin  is  not  active  in  alkaline  or  neu- 
tral solutions  but  requires  at  least  a  faint  acidity  before  it  can 
exert  its  power  to  dissolve  and  digest  proteins.  The  percentage 
of  hydrochloric  acid  facilitating  the  most  rapid  peptic  action  varies 
with  the  character  of  the  protein  acted  upon,  e.  g.,  0.08  per  cent  to 
0.1  per  cent  for  the  digestion  of  fibrin  and  0.25  per  cent  for  the  di- 
gestion of  coagulated  egg-white.  While  hydrochloric  acid  is  the 
acid  usually  employed  to  promote  artificial  peptic  proteolysis,  other 
acids,  organic  and  inorganic,  will  serve  the  same  purpose.  Acidity 
of  the  liquid  is  necessary  to  promote  the  activity  of  the  pepsin, 
but  the  acidity  need  not  necessarily  be  confined  to  hydrochloric 
acid. 

In  common  with  many  other  enzymes  pepsin  acts  best  at  about 
38°-40°  C.  and  its  digestive  power  decreases  as  the  temperature 
is  lowered,  the  enzyme  being  only  slightly  active  at  o°  C.  Its 
power  is  only  temporarily  inhibited  by  the  application  of  such 
low  temperatures,  however,  and  the  enzyme  regains  its  full  proteo- 
lytic power  upon  raising  the  temperature  to  40 °  C.  As  the  tempera- 
ture of  a  digestive  mixture  is  raised  above  400  C.  the  pepsin  grad- 
ually loses  its  activity  until  at  about  8o°-ioo°  C.  its  proteolytic 
power  is  permanently  destroyed. 

Our  ideas  regarding  the  nature  of  the  products  formed  in  the 
course  of  peptic  proteolysis  have  undergone  considerable  revision 
in  recent  years.  The  former  view  that  these  products  included 
only  acid  albuminate  (acid  metaprotein),  proteoses  and  peptones 
is  no  longer  tenable.  From  the  investigations  of  numerous  ob- 
servers we  have  learned  that  artificial  gastric  digestion  if  permitted 
to  proceed  for  a  sufficiently  long  period  will  yield,  in  addition  to 
proteoses  and  peptones,  a  long  list  of  protein  cleavage  products 
which  are  crystalline  in  character,  including  leucine,  tyrosine,  alanine, 
phenylalanine,  aspartic  acid,  glutamic  acid,  proline,  leucinimide, 
valine  and  lysine.     A  similar  group  of  substances  may  result  from 


GASTRIC    DIGESTION.  I  2  I 

the  action  of  the  enzyme  trypsin  (see  p.  141).  The  relative  amounts 
of  proteoses,  peptones  and  crystalline  substances  formed  depends 
to  a  great  extent  upon  the  character  of  the  protein  undergoing 
digestion,  e.  g.,  a  greater  proportion  of  proteoses  results  from  the 
digestion  of  fibrin  than  from  the  digestion  of  coagulated  egg-white. 
We  must  not  be  led  into  the  error  of  thinking  that  the  large  num- 
ber of  protein  cleavage  products  just  mentioned  are  formed  in  the 
course  of  normal  gastric  digestion  within  the  animal  organism. 
They  are  formed  only  after  comparatively  long-continued  hydroly- 
sis. In  pancreatic  digestion,  however,  there  are  formed  even  under 
normal  conditions,  the  large  number  of  cleavage  products  to  which 
reference  has  been  made.  Peptic  proteolysis  therefore,  within  the 
animal  organism  differs  from  tryptic  proteolysis  (see  page  141)  in 
that  the  former  yields  larger  amounts  of  proteoses,  smaller  amounts 
of  peptones  and  no  considerable  quantity  of  crystalline  bodies  as 
end-products  in  the  brief  period  during  which  proteins  are  ordinarily 
subjected  to  gastric  digestion.  Prolonged  hydrolysis  with  gastric 
juice  does,  however,  yield  considerable  quantities  of  the  non-pro- 
tein end-products. 

Gastric  rennin,  the  second  enzyme  of  the  gastric  juice,  is  what  is 
known  as  a  milk  curdling  or  protein  coagulating  enzyme.  Rennin 
acts  upon  the  caseinogen  of  the  milk,  splitting  it  into  a  proteose- 
like  body  and  soluble  casein.  This  soluble  body,  in  the  presence 
of  calcium  salts,  combines  with  calcium,  forming  calcium  casein 
or  true  casein  which  is  insoluble  and  precipitates.  There  is  some 
uncertainty  regarding  the  reaction  to  litmus  in  which  gastric 
rennin  shows  the  greatest  activity.  It  is,  however,  said  to  be  active 
in  neutral,  alkaline  or  acid  solution.  However,  it  probably  pos- 
sesses its  greatest  activity  in  the  presence  of  a  slight  acid  reaction,  as 
would  naturally  be  expected.  It  is  especially  abundant  in  the 
gastric  mucosa  of  the  calf,  and  is  used  to  curdle  the  milk  used  in 
cheese  making.  Gastric  rennin  is  always  present  normally  in  the 
gastric  juice  but  in  certain  pathological  conditions  such  as  atrophy 
of  the  mucosa,  chronic  catarrh  of  the  stomach  or  in  carcinoma  it 
may  be  absent. 

The  theory  that  the  proteolytic  activity  and  the  milk  curdling 
property  of  the  gastric  juice  reside  in  a  single  molecule  is  causing 
much  controversy  at  the  present  time.  The  theory  was  originally 
advanced  by  the  Pawlow  school. 

Gastric  lipase,  the  third  enzyme  of  the  gastric  juice,  is  a  fat- 
splitting  enzyme.     It  possesses  but  slight  activity  when  the  gastric 


122  PHYSIOLOGICAL    CHEMISTRY. 

juice  is  of  normal  acidity,  but  evinces  its  action  principally  at  such 
times  as  a  gastric  juice  of  low  acidity  is  secreted  either  from 
physiological  or  pathological  cause.  The  digestion  of  fat  in  the 
stomach  is,  however,  at  most,  of  but  slight  importance  as  com- 
pared with  the  digestion  of  fat  in  the  intestine  through  the  action  of 
the  lipase  of  the  pancreatic  juice  (see  page  143). 

PREPARATION  OF  AN  ARTIFICIAL  GASTRIC  JUICE. 

Dissect  the  mucous  membrane  of  a  pig's  stomach  from  the  mus- 
cular portion  and  discard  the  latter.  Divide  the  mucous  membrane 
into  two  parts  (4/5  and  1/5).  Cut  up  the  larger  portion,  place 
it  in  a  large-sized  beaker  with  0.4  per  cent  hydrochloric  acid  and 
keep  at  38°-40°  C.  for  at  least  24  hours.  Filter  off  the  residue, 
consisting  principally  of  nuclein  and  anti-albumid,  and  use  the 
filtrate  as  an  artificial  gastric  juice.  This  filtrate  contains  pepsin, 
rennin  and  the  products  of  the  digestion  of  the  stomach  tissue, 
i.  e.,  acid  metaprotein  (acid  albuminate),  proteoses,  peptones,  etc. 

Preparation    of    a    Glycerol    Extract    of    Pig's    Stomach. 

Take  the  one-fifth  portion  of  the  mucous  membrane  of  the  pig's 
stomach  not  used  in  the  preparation  of  the  artificial  gastric  juice, 
cut  it  up  very  finely,  place  it  in  a  small-sized  beaker  and  cover  the 
membrane  with  glycerol.  Stir  frequently  and  allow  to  stand  at 
room  temperature  for  at  least  24  hours.  The  glycerol  will  extract 
the  pepsinogen.  Separate,  with  a  pipette  or  by  other  means,  the 
glycerol  from  the  pieces  of  mucous  membrane  and  use  the  glycerol 
extract  as  required  in  the  later  experiments. 

Products    of    Gastric    Digestion. 

Into  the  artificial  gastric  juice,  prepared  as  above  described, 
place  the  protein  material  (fibrin,  coagulated  egg-white,  or  lean 
beef)  provided  for  you  by  the  instructor,  add  0.4  per  cent  hydro- 
chloric acid  as  suggested  by  the  instructor  and  keep  the  digestion 
mixture  at  400  C.  for  2  to  3  days.  Stir  frequently  and  keep  free 
hydrochloric  acid  present  in  the  solution  (for  tests  for  free  hydro- 
chloric acid  see  p.  123). 

The  original  protein  has  been  digested  and  the  solution  now  con- 
tains the  products  of  peptic  proteolysis,  i.  e.,  acid  metaprotein  (acid 
albuminate),  proteoses,  peptones,  etc.  The  insoluble  residue  may 
include  nuclein  and  anti-albumid.     Filter  the  digestive  mixture  and 


GASTRIC    DIGESTION.  I  23 

after  testing  for  free  hydrochloride  acid  neutralize  the  filtrate  with 
potassium  hydroxide  solution.  If  any  of  the  acid  metaprotein  (acid 
albuminate)  is  still  untransformed  into  proteoses  it  will  precipitate 
upon  neutralization.  If  any  precipitate  forms  heat  the  mixture 
to  boiling,  and  filter.  If  no  precipitate  forms  proceed  without  fil- 
tering. 

We  now  have  a  solution  containing  a  mixture  consisting  princi- 
pally of  proteoses  and  peptones.  Separate  and  identify  the  pro- 
teoses and  peptones  according  to  the  directions  given  on  pages  114 
and  115. 

Tests  for  Free  and  Combined  HC1. 

These  tests  are  made  with  a  class  of  reagents  known  as  indicators, 
so-called  because  they  serve  to  indicate  the  nature  of  the  reaction  of 
a  solution.  These  indicators  are  weak  acids  or  bases  and  are  but 
slightly  dissociable.  The  dissociation,  with  the  formation  of  the 
colored  ion,  forms  the  basis  for  the  color  reaction. 

Examine  each  of  the  following  solutions  by  means  of  the  tests 
given  below  and  report  the  results  in  a  form  similar  to  the  chart 
given  on  page  125:  (1)  0.2  per  cent  free  hydrochloric  acid.  (2) 
0.05  per  cent  free  hydrochloric  acid.  (3)  0.01  per  cent  free  hy- 
drochloric acid.  (4)  0.05  per  cent  combined  hydrochloric  acid. 
(5)  1  per  cent  lactic  acid.  (6)  Equal  volumes  of  0.2  per  cent  free 
hydrochloric  acid  and  1  per  cent  lactic  acid.  (7)  1  per  cent  potas- 
sium hydroxide. 

1.  Dimethyl-amino-azobenzene  (or  Topfer's  Reagent),1 

N(CH3)2-C6H4-N  =  N-C6H5. 

Place  1-2  drops  of  the  reagent  in  the  solution  to  be  tested.  Free 
mineral  acid  (hydrochloric  acid)  is  indicated  by  the  production  of  a 
pinkish-red  color.  If  free  acid  is  absent  a  yellow  color  ordinarily 
results. 

2.  Giinzberg's  Reagent.2 — Place  1-2  drops  of  the  reagent  in  a 
small  porcelain  evaporating  dish  and  carefully  evaporate  to  dryness 
over  a  low  flame.  Insert  a  glass  stirring  rod  into  the  mixture  to  be 
tested  and  draw  the  moist  end  of  the  rod  through  the  dried  reagent. 
Warm  again  gently  and  note  the  production  of  a  purplish-red 
color  in  the  presence  of  free  hydrochloric  acid. 

lTo  prepare  Topfer's  reagent  dissolve  0.5  gram  of  di-methyl-amino-azobenzene 
in  100  c.c.  of  95  per  cent  alcohol. 

"Giinzberg's  reagent  is  prepared  by  dissolving  2  grams  of  phloroglucin  and  1 
gram  of  vanillin  in  100  c.c.  of  95  per  cent  alcohol. 


124  PHYSIOLOGICAL    CHEMISTRY. 

3.  Boas'  Reagent.1 — Perform  this  test  in  the  same  manner  as 
2,  p.  123.  Free  hydrochloric  acid  is  indicated  by  the  production 
of  a  rose-red  color,  which  becomes  less  pronounced  on  cooling. 

4.  Congo  Red,2 

NH,  SO,Na 


'x  A/'  N=N<_><__>N=N  'x 
S08Na  NH2 

Conduct  this  test  according  to  the  directions  given  under  1  or  2, 
page  123.  A  blue  color  indicates  free  hydrochloric  acid,  a  violet 
color  indicates  an  organic  acid  and  a  brown  color  indicates  com- 
bined hydrochloric  acid.  Congo  red  paper,  made  by  immersing 
ordinary  filter  paper  in  the  indicator  and  subsequently  drying,  may 
be  used  in  this  test. 

5.  Tropaeolin  OO,3 

NH(C6HB)  -C6H4-N  =  N-C6H4-S03Na. 

Place  2  drops  of  the  solution  to  be  tested  and  1  drop  of  the  indicator 
in  an  evaporating  dish  and  evaporate  to  dryness  over  a  low  flame. 
The  formation  of  a  reddish-violet  color  indicates  free  hydrochloric 
acid. 

This  test  may  also  be  conducted  in  the  same  manner  as  2,  page 
123. 

6.  Phenolphthalein,4 

C6H4OH 

/ 
C-C6H4OH 

/"\ 

C6H4         0 

\/ 

C 

\ 

0 

Add  the  indicator  directly  to  the  solution,  or  apply  the  test  ac- 

1  Boas'  reagent  is  prepared  by  dissolving  5  grams  of  resorcin  and  3  grams  of 
sucrose  in  100  c.c.  of  95  per  cent  alcohol. 

-  This  indicator  is  prepared  by  dissolving  0.5  gram  of  congo  red  in  90  c.c.  of 
water  and  adding  10  c.c.  of  95  per  cent  alcohol. 

3  Prepared  by  dissolving  0.05  gram  of  tropaeolin  OO  in  100  c.c.  of  50  per  cent 
alcohol. 

*  This  indicator  is  prepared  by  dissolving  1  gram  of  phenolphthalein  in  100  c.c 
of  95  per  cent  alcohol. 


GASTRIC    DIGESTION. 


125 


cording  to  the  directions  given  under  2  on  page  123.  This  indicator 
serves  to  denote  the  total  acidity  since  it  is  acted  upon  by  free  min- 
eral acids,  combined  acids,  organic  acids  and  acid  salts.  A  red 
color  indicates  the  presence  of  an  alkali  and  the  indicator  is  colorless 
in  the  presence  of  a  neutral  or  acid  reaction.  This  indicator  is 
unsatisfactory  in  the  presence  of  ammonia. 
7.  Sodium  Alizarin  Sulphonate,1 

CO  (OH), 

/      \        / 
CGH4  CGH 

\      /        \ 

CO  SOJSTa 


This  indicator  may  be  used  directly  in  the  solution  to  be  tested,  or 
the  test  may  be  applied  as  2,  page  123.  It  serves  to  indicate  all  acid 
reactions  except  those  due  to  combined  acids.  A  reddish-violet 
color  indicates  an  alkaline  reaction,  while  a  yellow  color  indicates 
an  acid  reaction  due  to  a  free  mineral  acid,  an  organic  acid  or  an 
acid  salt. 

Report  the  results  of  your  tests  tabulated   in  the   form  given 
below : 


Name  of  Indicator. 

Solutions  Examined. 

0.2  $ 
HC1. 

0.05  % 
HU. 

O.OI   <& 

HC1. 

0.0s  $  1     1  a       EciuaJ  vols. 

Combined      Lactic     1      o.s*HU             x* 
HO.           Acid.        T    an.d  '/<           KOH. 
Lactic  Acid. 

Topfer's  Reagent. 
Giinzberg's  Reagent. 

Boas'  Reagent. 

Congo  Red. 

Tropseolin  OO. 
Phenolphthalein. 

j 

Alizarin. 

1 

GENERAL    EXPERIMENTS    ON    GASTRIC 
DIGESTION. 

I.  Conditions  Essential  for  the  Action  of  Pepsin. — Prepare 
four  test-tubes  as  follows : 

(a)   Five  c.c.  of  pepsin  solution. 

1  Prepare  this  indicator  by  dissolving  i  gram  of  sodium  alizarin  sulphonate  in 
ioo  c.c.  of  water. 


126  PHYSIOLOGICAL    CHEMISTRY. 

(b)  Five  c.c.  of  0.4  per  cent  hydrochloric  acid. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

(d)  Two  or  three  c.c.  of  pepsin  solution  and  2-3  c.c.  of  0.5  per 
cent  sodium  carbonate  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  place  them 
on  the  water-bath  at  400  C.  for  one-half  hour,  carefully  noting  any 
changes  which  occur.1  Now  combine  the  contents  of  tubes  (a) 
and  (b)  and  see  if  any  further  change  occurs  after  standing  at  400 
C.  for  15-20  minutes.  Explain  the  results  obtained  from  these 
five  experiments. 

2.  Influence  of  Different  Temperatures. — In  each  of  four  test- 
tubes  place  5  c.c.  of  pepsin-hydrochloric  acid  solution.  Immerse 
one  tube  in  cold  water  from  the  faucet,  keep  a  second  tube  at  room 
temperature  and  place  a  third  on  the  water-bath  at  400  C.  Boil 
the  contents  of  the  fourth  tube  for  a  few  moments,  then  cool  and 
also  keep  it  at  40 °  C.  Into  each  tube  introduce  a  small  piece  of 
fibrin  and  note  the  progress  of  digestion.  In  which  tube  does  the 
most  rapid  digestion  occur?     Explain  this. 

3.  The  Most  Favorable  Acidity. — Prepare  three  tubes  as 
follows : 

(a)  Five  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution. 

(b)  Two  or  three  c.c.  of  0.2  per  cent  hydrochloric  acid  -f-  1  c.c. 
of  concentrated  hydrochloric  acid  -j-  5  c.c.  of  pepsin  solution. 

(c)  One  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution 
-f-  5  c.c.  of  water. 

Introduce  a  small  piece  of  fibrin  into  each  tube,  keep  them  at 
400  C,  and  note  the  progress  of  digestion.  In  which  degree  of 
acidity  does  the  fibrin  digest  the  most  rapidly? 

4.  Differentiation  Between  Pepsin  and  Pepsinogen. — Prepare 
five  tubes  as  follows : 

(a)  Few  drops  of  glycerol  extract  of  pepsinogen  -f-  2-3  c.c.  of 
water. 

(b)  Few  drops  of  glycerol  extract  of  pepsinogen  -f-  5  c.c.  of  0.2 
per  cent  hydrochloric  acid. 

1  Digestion  of  fibrin  in  a  pepsin-hydrochloric  acid  solution  is  indicated  first  by 
a  swelling  of  the  protein  due  to  the  action  of  the  acid,  and  later  by  a  disintegra- 
tion and  dissolving  of  the  fibrin  due  to  the  action  of  the  pepsin-hydrochloric  acid. 
If  uncertain  at  any  time  whether  digestion  has  taken  place,  the  solution  under 
examination  may  be  filtered  and  the  biuret  test  applied  to  the  filtrate.  A  positive 
reaction  will  signify  the  presence  of  acid  metaprotein  (acid  albuminate),  pro- 
teoses (albumoses)  or  peptones,  the  presence  of  any  one  of  which  would  indi- 
cate that  digestion  has  taken  place. 


GASTRIC    DIGESTION.  I  2J 

(c)  Few  drops  of  glycerol  extract  of  pepsinogen  -f-  5  c.c.  of  0.5 
per  cent  sodium  carbonate. 

(d)  Two  or  three  c.c.  of  pepsin  solution  -f-  2-3  c.c.  of  1  per  cent 
sodium  carbonate. 

(<?)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  1 
per  cent  sodium  carbonate. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube,  keep 
the  five  tubes  at  40 °  C.  for  one-half  hour  and  observe  any  changes 
which  may  have  occurred.  To  (a)  add  an  equal  volume  of  0.4 
per  cent  hydrochloric  acid,  neutralize  (c),  (d)  and  (e)  with  hy- 
drochloric acid  and  add  an  equal  volume  of  0.4  per  cent  hydro- 
chloric acid.  Place  these  tubes  at  400  C.  again  and  note  any  fur- 
ther changes  which  may  occur.  What  contrast  do  we  find  in  the 
results  from  the  three  last  tubes?     Why  is  this  so? 

5.  Comparative  Digestive  Power  of  Pepsin  with  Different 
Acids. — Prepare  a  series  of  tubes  each  containing  one  of  the  follow- 
ing acids  :  0.5  per  cent  acetic,  lactic,  oxalic,  salicylic,  tannic  and  buty- 
ric, and  0.2  per  cent  hydrochloric,  sulphuric,  nitric,  arsenious,  and 
combined  hydrochloric.  To  each  acid  add  a  few  drops  of  the  gly- 
cerol extract  of  pig's  stomach  and  a  small  piece  of  fibrin.  Shake 
well,  place  at  400  C.  and  note  the  progress  of  digestion.  In  which 
tubes  does  the  most  rapid  digestion  occur? 

6.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of  tubes 
and  into  each  tube  introduce  4  c.c.  of  pepsin-hydrochloric  acid  so- 
lution and  Y-2  c.c.  of  one  of  the  chemicals  listed  in  Experiment  18 
under  Salivary  Digestion,  page  59.  Introduce  a  small  piece  of 
fibrin  into  each  of  the  tubes  and  keep  them  at  40 °  C.  for  one-half 
hour.  Note  the  variations  in  the  progress  of  digestion.  Where 
has  the  least  rapid  digestion  occurred? 

7.  Sahli's  Desmoid  Reaction. — This  is  a  method  for  testing  gas- 
tric function  without  using  the  stomach  tube.  The  underlying 
principle  of  the  test  is  the  fact  that  raw  catgut  may  be  digested 
in  gastric  juice  but  is  entirely  indigestible  in  pancreatic  juice.  The 
test  is  made  as  follows :  A  methylene  blue  pill  is  introduced  into 
a  small  rubber  bag  and  the  mouth  of  the  bag  subsequently  tied  with 
catgut.1     The  small  bag  is  then   ingested  immediately  after  the 

1  About  0.05  gram  of  methylene  blue  is  mixed  with  sufficient  ext.  glycyrrJiizcc 
to  form  a  pill  about  3-4  mm.  in  diameter.  The  pill  is  then  placed  in  the  center 
of  a  square  piece  of  thin  rubber  dam  and  a  little  bag-like  receptacle  constructed 
by  a  twisting  movement.  The  neck  of  the  bag  is  then  closed  by  wrapping  three 
turns  of  catgut  about  it.  The  most  satisfactory  catgut  to  use  is  number  00  raw 
catgut  which  has  previously  been  soaked  in  water  until  soft.  When  ready  for 
use  the  bag  should  sink  instantly  when  placed  in  water  and  be  water-tight. 


128  PHYSIOLOGICAL    CHEMISTRY. 

mid-day  meal  and  the  urine  examined  5,  7,  9  and  18-20  hours  later 
for  methylene  blue.  If  methylene  blue  is  present  in  appreciable 
quantity,  it  will  impart  to  the  urine  a  greenish-blue  color.  If  not 
present  in  sufficient  amount  to  impart  this  color  the  urine  should  be 
boiled  with  )4  its  volume  of  glacial  acetic  acid  whereupon  a  green- 
ish-blue color  results  if  the  chromogen  of  methylene  blue  is  present. 
This  contingency  seldom  arises,  however,  inasmuch  as  in  most  cases 
of  uncolored  urine  it  will  be  found  that  the  rubber  bag  has  passed 
through  the  stomach  unopened.  If  the  methylene  blue  is  found  in 
the  urine  inside  of  18-20  hours  a  satisfactory  gastric  function  is  in- 
dicated. 

8.  Testing  the  Motor  and  Functional  Activities  of  the 
Stomach. — This  test  is  performed  the  same  as  Experiment  19  under. 
Salivary  Digestion,  page  60.  If  the  experiment  was  carried  out 
under  salivary  digestion  it  will  not  be  necessary  to  repeat  it  here. 

9.  Influence  of  Bile. — Prepare  five  tubes  as  follows : 

(a)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -f-  Y2—1  c.c.  of 
bile. 

(b)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -f-  1-2  c.c. 
of  bile. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -j-  2-3  c.c.  of 
bile. 

(d)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -f-  5  c.c.  of  bile. 
(<?)    Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin.  Keep  the 
tubes  at  400  C.  and  note  the  progress  of  digestion.  Does  the  bile 
exert  any  appreciable  influence?    How? 

10.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of 
five  tubes  as  follows : 

(a)  Five  c.c.  of  fresh  milk  -f-  0.2  per  cent  hydrochloric  acid  (add 
slowly  until  precipitate  forms). 

(&)    Five  c.c.  of  fresh  milk  -(-  5  drops  of  rennin  solution. 

(c)  Five  c.c.  of  fresh  milk  -j-  10  drops  of  0.5  per  cent  sodium 
carbonate  solution. 

(d)  Five  c.c.  of  fresh  milk  -f-  10  drops  of  a  saturated  solution 
of  ammonium  oxalate. 

(e)  Five  c.c.  of  fresh  milk  -j-  5  drops  of  0.2  per  cent  hydrochloric 
acid.  Now  to  each  of  the  tubes  (c),  (d)  and  (e)  add  5  drops  of 
rennin  solution.  Place  the  whole  series  of  five  tubes  at  400  C.  and 
after  10-15  minutes  note  what  is  occurring  in  the  different  tubes. 
Give  a  reason  for  each  particular  result. 


GASTRIC    DIGESTION.  \  2<j 

ii.  Tests  for  Lactic  Acid,     (a)  Uffelmann's  Reaction. — To  a 

small  quantity  of   Uffelmann's  reagent1    in   a  tesl  tube  add  a 
drops  of  a  lactic  acid  solution.     The  amethyst-blue  color  of   the 
reagent  is  displaced  by  a  straw  yellow.     Other  organic  acids  give  a 
similar  reaction.     Mineral  acids  such  as  hydrochloric  acid  discharge 
the  blue  coloration  leaving  a  colorless  solution. 

(b)  Ferric  Chloride  Test. — Place  10  c.c.  of  very  dilute  ferric 
chloride  in  each  of  five  tubes.  To  the  first  add  2  c.c.  of  0.2  per 
cent  hydrochloric  acid,  to  the  second  2  c.c.  of  10  per  cent  alcohol, 
to  the  third  2  c.c.  of  2  per  cent  sucrose,  to  the  fourth  2  c.c.  of 
lactic  acid  and  to  the  fifth  2  c.c.  of  peptone  solution. 

It  is  evident  from  the  results  obtained  that  neither  of  the  tests 
given  above  is  satisfactory  for  the  detection  of  lactic  acid  in  the 
presence  of  other  substances  such  as  we  find  in  the  gastric  contents. 

A  satisfactory  deduction  regarding  the  presence  of  lactic  acid  can 
only  be  made  after  extracting  the  gastric  contents  with  ether,  evapor- 
ating the  ether  extract  to  dryness  and  dissolving  the  residue  in  water. 
This  residue  will  not  contain  any  of  the  contaminations  which  in- 
terfered with  the  simple  tests  as  tried  above,  and  therefore  if  either 
of  the  tests  is  now  tried  on  the  dissolved  residue  of  the  ether  extract 
we  may  form  an  accurate  conclusion  regarding  the  presence  of  lactic 
acid. 

(c)  Hopkins'  Thiophene  Reaction. — Place  about  5  c.c.  of  concen- 
trated sulphuric  acid  in  a  test-tube  and  add  one  drop  of  a  saturated 
solution  of  cupric  sulphate.2  Introduce  a  few  drops  of  the  solution 
to  be  tested,  shake  the  tube  well  and  immerse  it  in  the  boiling 
water  of  a  beaker-water-bath  for  one  or  two  minutes.  Now  remove 
the  tube,  cool  it  under  running  water,  add  2-3  drops  of  a  dilute 
alcoholic  solution3  of  thiophene,  C4H4S,  from  a  pipette,  replace 
the  tube  in  the  beaker  and  carefully  observe  any  color  change  which 
may  occur.  Lactic  acid  is  indicated  by  the  appearance  of  a  bright 
cherry-red  color  which  forms  rapidly.  This  color  may  be  made 
more  or  less  permanent  by  cooling  the  tube  as  soon  as  the  color  is 
produced.  Excess  of  thiophene  produces  a  deep  yellow  or  brown 
color  with  sulphuric  acid.  The  test  is  not  wholly  specific  though 
the  author  claims  it  to  be  more  so  than  Uffelmann's  reaction. 

12.   Qualitative  Analysis  of  Stomach  Contents. — Take  100  c.c. 

1  Uffelmann's  reagent  is  prepared  by  adding  ferric  chloride  solution  to  a  1  per 
cent  solution  of  carbolic  acid  until  an  amethyst-blue  color  is  obtained. 
■This   is  added  to  catalyze  the  oxidation   which   follows. 
s  About   10-20  drops   in   100  c.c.  of  95  per  cent  alcohol. 


130 


PHYSIOLOGICAL    CHEMISTRY. 


of  stomach   contents   and   analyze  it   according   to   the    following 
scheme : 

Stomach    Contents. 
Filter  and  test  the  filtrate  for  free  hydrochloric  acid. 


Filtrate  I. 
Divide  into  two  parts. 


Residue. 
Discard  after  making  a  micro- 
scopical examination. 


Filtrate  II. 
One-fifth  portion. 
Test  for : 

(a)  Pepsin. 

(b)  Bile  (see  p.  154). 

(c)  Starch. 

(d)  Dextrin. 


Filtrate  III. 
P'our-fifths  portion. 

Neutralize  carefully ;  any  precipitate  is  acid 
metaprotein  (acid  albuminate).  If  a  pre- 
cipitate forms  filter  and  divide  the  filtrate 
into  two  parts.  If  no  precipitate  forms 
divide  the  solution  into  two  parts  without 
filtering. 


Filtrate  IV. 
Two-thirds  portion. 
Heat  to  boiling  to  remove  coagulable 
proteins.     If  any  precipitate  forms 
filter  it  off;  if  there  is  no  precipi- 
tate proceed  directly  with  the  tests. 
Test  for : 

(a)  Sugar.       (Differentiate     between 

various    sugars    by    the    use    of 
scheme  on  page  51.) 

(b)  Proteoses. 

(c)  Peptones. 


Filtrate  V. 
One-third  portion. 
Test  for : 

(a)  Lactic  acid. 

(b)  Gastric  rennin. 
(r)   Salivary  amylase. 


the 
the 


CHAPTER  VII. 


FATS. 


Fats  occur  very  widely  distributed  in  the  plant  and  animal  king- 
doms, and  constitute  the  third  general  class  of  food  stuffs.  In  plant 
organisms  they  are  to  be  found  in  the  seeds,  roots  and  fruit,  while 
each  individual  tissue  and  organ  of  an  animal  organism  contains 
more  or  less  of  the  substance.  In  the  animal  organism  fats  are 
especially  abundant  in  the  bone  marrow  and  adipose  tissue.  They 
contain  the  same  elements  as  the  carbohydrates,  i.  e.,  carbon,  hydro- 
gen and  oxygen,  but  the  oxygen  is  present  in  smaller  percentage 
than  in  the  carbohydrates  and  the  hydrogen  and  oxygen  are  not 
present  in  the  proportion  to  form  water. 

Fig.  35. 


Seef    Fat.      (Long.) 


Chemically  considered  the  fats  are  esters1  of  the  tri-atomic  alco- 
hol, glycerol,  and  the  mono-basic  fatty  acids.  In  the  formation  of 
these  fats  three  molecules  of  water  result.  This  water  may  arise 
in  either  of  two  ways.  First,  by  the  replacement  of  the  H  of 
each  of  the  OH  groups  of  glycerol  by  a  fatty  acid  radical,  giving 
the  following  formula  in  which  R,  R'  and  R"  represent  fatty  acid 
radicals, 

1  An  ester  is  an  ethereal  salt  consisting  of  an  organic  radical  united  with  the 
residue  of  an  inorganic  or  organic  acid. 

131 


132  PHYSIOLOGICAL    CHEMISTRY. 

CH2OR 
CH  OR' 
CH2OR". 

Second,  by  the  replacement  of  the  H's  of  the  carboxyl  groups  of 

the  three  fatty  acid  molecules  by  the  glycerol  radical,  thus  yielding 

the  following  type  of  formula  in  which  R  represents  the  glycerol 

radical, 

OOCH31C15 

/ 
R-OOCH31C15 

\ 

OOCH3A5. 

Of  these  two  processes  the  second  is  the  more  logical  procedure 
from  the  standpoint  of  the  ionic  theory.  The  three  fatty  acid 
radicals  entering  into  the  structure  of  a  neutral  fat  may  be  the 
radicals  of  the  same  fatty  acid  or  they  may  consist  of  the  radicals 
of  three  different  fatty  acids. 

By  hydrolysis  of  a  neutral  fat,  i.  e.,  by  the  addition  to  the  mole- 
cule of  those  elements  which  are  eliminated  in  the  formation  of 
the  fat  from  glycerol  and  fatty  acid,  it  may  be  resolved  into  its 
component  parts,  i.  e.,  glycerol  and  fatty  acid.  In  the  case  of  tri- 
palmitin  the  following  would  be  the  reaction: 

C3H5(0-C15H31CO)3+3H20  =  C3H5(OH)3+3(C15H31COOH). 

Tri-palmitin.  Glycerol.  Palmitic  acid. 

This  process  is  called  saponification  and  may  be  produced  by  boil- 
ing with  alkalis ;  by  the  action  of  steam  under  pressure ;  by  long- 
continued  contact  with  air  and  light;  by  the  action  of  certain  bac- 
teria and  by  fat-splitting  ezymes  or  lipases,  e.  g.,  pancreatic  lipase 
(see  page  143).  The  cells  forming  the  walls  of  the  intestines  evi- 
dently possess  the  peculiar  property  of  synthesizing  the  glycerol  and 
fatty  acid  thus  formed  so  that  after  absorption  these  bodies  appear 
in  the  blood  not  in  their  individual  form  but  as  neutral  fats.  This 
synthesis  is  similar  to  that  enacted  in  the  absorption  of  protein 
material  where  the  peptones  are  synthesized  into  albumin  in  the 
act  of  absorption. 

The  principal  animal  fats  with  which  we  have  to  deal  are  stearin, 
palmitin,  olcin  and  butyrin.     Such  less  important  forms  as  laurin 


FATS.  133 

and  myristin  may  occur  abundantly  in  plant  organisms.  The  gen- 
erally accepted  system  of  nomenclature  for  these   fats  is  to  apply 

the  prefix  "  tri  "  in  each  case  (c.  g.,  /n-palmitin  )  since  three  fatty 
acid  radicals  are  contained  in  the  neutral   fat  molecule. 

Fats  occur  ordinarily  as  mixtures  of  several  individual  fats. 
For  example,  the  fat  found  in  animal  tissues  is  a  mixture  of  tri- 
olein, tri-palmitin  and  tri-stearin,  the  percentage  of  any  one  of 
these  fats  present  depending  upon  the  particular  species  of  animal 
from  whose  tissue  the  fat  was  derived.  Thus  the  ordinary  mutton 
fat  contains  more  tri-stearin  and  less  tri-olein  than  the  pork  fat. 
Human  fat  contains  from  67  per  cent  to  85  per  cent  of  tri-olein 
and  according  to  Benedict  and  Osterberg,  upon  analysis  yields 
76.08  per  cent  of  carbon  and  11.78  per  cent  of  hydrogen. 

Pure  neutral  fats  are  odorless,  tasteless  and  generally  colorless. 
They  are  insoluble  in  the  ordinary  protein  solvents  such  as  water, 
salt  solutions  and  dilute  acids  and  alkalis  but  are  very  readily 
soluble  in  ether,  benzene,  chloroform  and  boiling  alcohol.  The 
neutral  fats  are  non-volatile  substances  possessing  a  neutral  reac- 
tion. If  allowed  to  remain  in  contact  with  the  air  for  a  sufficient 
length  of  time  they  become  yellow  in  color,  assume  an  acid  reaction 
and  are  said  to  be  rancid.  The  neutral  fats  may  be  crystallized, 
some  of  them  with  great  facility.  The  crystalline  forms  of  some 
of  the  more  common  fats  are  reproduced  in  Figs.  35,  36  and  37 
on  pages  131,  134  and  136.  Each  individual  fat  possesses  a  specific 
melting-  or  boiling-point  (according  to  whether  the  body  is  solid 
or  fluid  in  character)  and  this  property  of  melting  or  boiling  at  a 
definite  temperature  may  be  used  as  a  means  of  differentiation  in 
the  same  way  as  the  coagulation  temperature  (see  page  242)  is 
used  for  the  differentiation  of  coagulable  proteins.  When  shaken 
with  water,  or  a  solution  of  albumin,  soap,  or  acacia,  the  liquid 
fats  are  finely  divided  and  assume  a  condition  known  as  an  emul- 
sion. The  emulsion  with  water  is  transitory,  while  the  emulsions 
with  soap,  acacia,  or  albumin,  are  permanent. 

The  fat  ingested  continues  essentially  unaltered  until  it  reaches 
the  intestines  where  it  is  acted  upon  by  pancreatic  lipase  (steapsiu) 
the  fat-splitting  enzyme  of  the  pancreatic  juice  (see  page  143), 
and  glycerol  and  fatty  acid  are  formed  from  a  large  portion  of 
the  fat.  Part  of  the  fatty  acid  thus  formed  is  dissolved  in  the 
bile  and  absorbed  while  the  remainder  unites  with  the  alkalis  of 
the  pancreatic  juice  and  forms  soluble  soaps.  These  soaps  may 
further  act  to  produce  an  emulsion  of  the  remaining  fat  and  thus 


134  PHYSIOLOGICAL    CHEMISTRY. 

aid  in  its  absorption.  That  bile  is  of  assistance  in  the  absorption 
of  fat  is  indicated  by  the  increase  of  fat  in  the  feces  when  for  any 
reason  bile  does  not  pass  into  the  intestines. 

The  fat  distributed  throughout  the  animal  body  is  formed  partly 
from   the   ingested    fat   and    partly    from   carbohydrates   and    the 

Fig.  36. 


ttafft 

^w    i< 

1    ,"'• 

[  \  '    \ 

®m  ^S<A 

!  ' 

BSflilll 

HH 

^m 

'      0  'll|li''  l! 

Pill 

H6I 
■"■■■ 

^m         f 

1 

*v  >?$C  0.1' 

IB 

Bc^  -• 

SKKM 

^tagBpS,'- 

%'M 

'&/  7m 

|fe 

Mutton   Fat.      (Long.) 


"  carbon  moiety  "  of  protein  material.  The  formation  of  adipocere 
and  the  occurrence  of  fatty  degeneration  are  sometimes  given  as 
proofs  of  the  formation  of  fat  from  protein.  This  is  questioned 
by  many  investigators.  Rather  more  satisfactory  and  direct  proof 
of  the  formation  of  fat  from  protein  material  has  been  obtained 
by  Hofmann  in  experimentation  with  fly-maggots.  The  normal 
content  of  fat  in  a  number  of  maggots  was  determined  and  later 
the  fat  content  of  others  which  had  developed  in  blood  (84  per 
cent  of  the  solid  matter  of  blood  plasma  is  protein  material)  was 
determined.  The  fat  content  was  found  to  have  increased  700  to 
1 100  per  cent  as  a  result  of  the  diet  of  blood  proteins.  The  cele- 
brated experiments  of  Pettenkofer  and  Voit,  however,  have  fur- 
nished what  is,  perhaps,  the  most  substantial  positive  evidence  of 
the  formation  of  fat  from  protein.  These  investigators  fed  dogs 
large  amounts  of  lean  meat,  daily,  and  through  subsequent  urinary 
and  fecal  examinations  were  enabled  to  account  for  only  part  of 
the  ingested  carbon,  although  obtaining  a  satisfactory  nitrogen 
balance.  The  discrepancy  in  the  carbon  balance  was  explained 
upon  the  theory  that  the  protein  of  the  ingested  meat  had  been 
split  into  a  nitrogenous  and  a  non-nitrogenous  portion  in  the  organ- 
ism, and  that  the  non-nitrogenous  portion,  the  so-called  "  carbon 


FATS.  I  3  5 

moiety"  of  the  protein,  had  been  subsequently  transformed  into 
fat  and  deposited  as  such  in  the  tissues  of  the  organism.  Some  in- 
vestigators are  not  inclined  to  accept  these  data  regarding  the  for- 
mation of  fat  from  protein  as  conclusive. 

The  latest  evidence  in  favor  of  the  formation  of  fat  from  pro- 
tein is  furnished  by  the  very  recently  reported  experiments  of 
Weinland.  This  investigator  worked  with  the  larva?  of  Calliphora,1 
these  larvae  being  rubbed  up  in  a  mortar"  with  Witte's  peptone  and 
water  to  form  a  homogeneous  mixture.  After  placing  these  mix- 
tures at  380  C.  for  24  hours  the  fat  content  was  found  to  have 
increased,  as  much  as  140  per  cent  in  some  instances.  The  active 
agency  in  this  transformation  of  fat  is  the  larval  tissue  since  the 
tissues  of  both  the  dead  and  living  larvae  possess  the  property. 
Data  are  given  from  control  tests  which  show  that  the  action  of 
bacteria  in  this  transformation  of  protein  was  excluded. 

Experiments  on  Fats. 

1.  Solubility. — Test  the  solubility  of  olive  oil  in  each  of  the 
ordinary  solvents  (see  page  23)  and  in  cold  alcohol,  hot  alcohol, 
chloroform,  ether,  and  carbon  tetrachloride. 

2.  Formation  of  a  Transparent  Spot  on  Paper. — Place  a  drop 
of  olive  oil  upon  a  piece  of  ordinary  writing  paper.  Note  the  trans- 
parent appearance  of  the  paper  at  the  point  of  contact  with  the  fat. 

3.  Reaction. — Try  the  reaction  of  fresh  olive  oil  to  litmus. 
Repeat  the  test  with  rancid  olive  oil.  What  is  the  reaction  of  a 
fresh  fat  and  how  does  this  reaction  change  upon  allowing  the 
fat  to  stand  for  some  time? 

4.  Formation  of  Acrolein. — To  a  little  olive  oil  in  a  mortar 
add  some  dry  potassium  bisulphate,  KHS04,  and  rub  up  thor- 
oughly. Transfer  to  a  dry  test-tube  and  cautiously  heat.  Note 
the  irritating  odor  of  acrolein.  The  glycerol  of  the  fat  has  been 
dehydrolyzed  and  acrylic  aldehyde  or  acrolein  has  been  produced. 
This  is  the  reaction  which  takes  place : 

CH2OH  CHO 

CH-OH    ->    CH  +  2PLO. 
CH2-OH  CH2 

Glycerol.  Acrolein. 

1  The  ordinary  "  blow-fly." 

2  Intact  larvse  were  used  in  some  experiments. 


136 


PHYSIOLOGICAL    CHEMISTRY. 


5.  Emulsification. —  (a)  Shake  up  a  drop  of  neutral1  olive  oil 
with  a  little  water  in  a  test-tube.  The  fat  becomes  finely  divided, 
forming  an  emulsion.  This  is  not  a  permanent  emulsion  since  the 
fat  separates  and  rises  to  the  top  upon  standing. 

(b)  To  5  c.c.  of  water  in  a  test-tube  add  2  or  3  drops  of  0.5 
per  cent  Na2C03.  Introduce  into  this  faintly  alkaline  solution  a 
drop  of  neutral  olive  oil  and  shake.  The  emulsion  while  not  per- 
manent is  not  so  transitory  as  in  the  case  of  water  free  from 
sodium  carbonate. 

(c)  Repeat  (b)  using  rancid  olive  oil.  What  sort  of  an  emul- 
sion do  you  get  and  why? 

(d)  Shake  a  drop  of  neutral  olive  oil  with  a  dilute  albumin 
solution.  What  is  the  nature  of  this  emulsion?  Examine  it  under 
the  microscope. 

6.  Fat  Crystals. — Dissolve  a  small  piece  of  lard  in  ether  in  a 
test-tube,  add  an  equal  volume  of  alcohol  and  allow  the  alcohol- 

Fig.  37. 


Pork  Fat. 


ether  mixture  to  evaporate  spontaneously.  Examine  the  crystals 
under  the  microscope  and  compare  them  with  those  reproduced  in 
Figs.  35,  36  and  37,  on  pages  131,  134  and  136. 

7.  Saponification  of  Bayberry  Tallow.2 — Fill  a  large  casserole 

1  Neutral  olive  oil  may  be  prepared  by  shaking  ordinary  olive  oil  with  a  10 
per  cent  solution  of  sodium  carbonate.  This  mixture  should  then  be  extracted 
with  ether  and  the  ether  removed  by  evaporation.  The  residue  is  neutral 
olive  oil. 

2  Bayberry  tallow  is  derived  from  the  fatty  covering  of  the  berries  of  the  wax 
myrtle.     It  is  therefore  frequently  called  "  myrtle  wax  "  or  "  bayberry  wax." 


FATS. 


137 


two-thirds  full  of  water  rendered  strongly  alkaline  with  solid  potas- 
sium hydroxide  (a  stick  one  inch  in  length).  Add  about  10  grams 
of  bayberry  tallow  and  boil,  keeping  the  volume  constanl  by  adding 
water  as  needed.  When  saponification  is  complete1  remove  25  c.c. 
of  the  soap  solution  for  use  in  Experiment  8  and  add  concentrated 
hydrochloric  acid  slowly  to  the  remainder  until  no  further  precipi- 
tate is  produced.2  Cool  the  solution  and  the  precipitate  of  free 
fatty  acid  will  rise  to  the  surface  and  form  a  cake.  In  this  instance 
the  fatty  acid  is  principally  palmitic  acid.  Remove  the  cake,  break 
it  into  small  pieces,  wash  it  with  water  by  clecantation  and  transfer 


Fig.    -8. 


Palmitic   Acid. 


to  a  small  beaker  by  means  of  95  per  cent  alcohol.  Heat  on  a 
water-bath  until  the  palmitic  acid  is  dissolved,  then  filter  through 
a  dry  filter  paper  and  allow  the  filtrate  to  cool  slowly  in  order  to 
obtain  satisfactory  crystals.  ^Trite  the  reactions  which  have  taken 
place  in  this  experiment. 

When  the  palmitic  acid  has  completely  crystallized  filter  off  the 
alcohol,  dry  the  crystals  between  filter  papers  and  try  the  tests 
given  in  Experiment  9,  p.  138. 

8.  Salting-out  Experiment. — To  25  c.c.  of  soap  solution,  pre- 
pared as  described  above,  add  solid  sodium  chloride  to  the  point 
of    saturation,    with    continual    stirring.      A    menstruum    is    thus 

1  Place  2  or  3  drops  in  a  test-tube  full  of  water.  If  saponification  is  complete 
the  products  will  remain  in  solution  and  no  oil  will  separate. 

2  Under  some  conditions  a  purer  product  is  obtained  if  the  soap  solution  is 
cooled   before  precipitating  the   fatty   acid. 


138 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  39. 


formed  in  which  the  soap  is  insoluble.     This  salting-out  process 
is  entirely  analogous  to  the  salting-out  of  proteins  (see  page  99). 
9.  Palmitic  Acid. —  (a)  Examine  the  crystals  under  the  micro- 
scope and  compare  them  with  those  shown  in  Fig.  38,  p.  137. 

(b)  Solubility. — Try  the  solubility 
of  palmitic  acid  in  the  same  solvents  as 
used  on  fats  (see  page  135). 

(c)  Melting-Point. — Determine  the 
melting-point  of  palmitic  acid  by  one  of 
the  methods  given  on  page  139. 

(d)  Formation  of  Transparent  Spot 
on  Paper. — Melt  a  little  of  the  fatty 
acid  and  allow  a  drop  to  fall  upon  a 
piece  of  ordinary  writing  paper.  How 
does  this  compare  with  the  action  of  a 
fat  under  similar  circumstances  ? 

(e)  Acrolein  Test. — Apply  the  test 
as  given  under  4,  page  135.  Explain 
the  result. 

10.  Saponification  of  Lard. — To 
25  grams  of  lard  in  a  flask  add  75 
c.c.  of  alcoholic-potash  solution  and 
warm  upon  a  water-bath  until  saponi- 
fication is  complete.  (This  point  is 
indicated  by  the  complete  solubility  of 
a  drop  of  the  solution  when  allowed  to 
fall  into  a  little  water.)  Now  trans- 
fer the  solution  from  the  flask  to  an 
evaporating'  dish  containing  about  100 
c.c.  of  water  and  heat  on  a  water-bath 
until  all  the  alcohol  has  been  driven 
off.  Precipitate  the  fatty  acid  with  hydrochloric  acid  and  cool  the 
solution.  Remove  the  fatty  acid  which  rises  to  the  surface,  neu- 
tralize the  solution  with  sodium  carbonate  and  evaporate  to  dryness. 
Extract  the  residue  with  alcohol,  remove  the  alcohol  by  evaporation 
upon  a  water-bath  and  on  the  residue  of  glycerol  thus  obtained 
make  the  tests  as  given  below. 

11.  Glycerol,     (a)   Taste. — What  is  the  taste  of  glycerol? 

(b)  Solubility. — Try  the  solubility  of  glycerol  in  water,  alcohol 
and  ether. 

(c)  Acrolein  Test. — Repeat  the  test  as  given  under  4,  page  135. 


Melting-Point   Apparatus. 


FATS.  139 

((/)  Borax  Fusion  Test. — Fuse  a  little  glycerol  on  a  platinum 
wire  with  some  powdered  borax  and  note  the  characteristic  green 
flame.     This  color  is  due  to  the  glycerol  ester  of  boric  acid. 

(e)  Fehling's  Test. — How  does  this  result  compare  with  the 
results  on  the  sugars? 

(/)  Solution  of  Cu(OH)2. — Form  a  little  cupric  hydroxide  by 
mixing  cupric  sulphate  and  potassium  hydroxide.  Add  a  little 
glycerol  to  this  suspended  precipitate  and  note  what  occurs. 

12.  Melting-Point  of  Fat.  First  Method. — Insert  one  of  the 
melting-point  tubes,  furnished  by  the  instructor,  into  the  liquid  fat 
and  draw  up  the  fat  until  the  bulb  of  the  tube  is  about  one-half 
full  of  the  material.  Then  fuse  one  end  of  the  tube  in  the  flame 
of  a  bunsen  burner  and  fasten  the  tube  to  a  thermometer  by  means 
of  a  rubber  band  in  such  a  manner  that  the  bottom  of  the  fat 
column  is  on  a  level  with  the  bulb  of  the  thermometer  (Fig.  39, 
page  138).  Fill  a  beaker  of  medium  size  about  two-thirds  full  of 
water  and  place  it  within  a  second  larger  beaker  which  also  contains 
water,  the  two  vessels  being  separated  by  pieces  of  cork.  Immerse 
the  bulb  of  the  thermometer  and  the  attached  tube  in  such  a  way 
that  the  bulb  is  about  midway  between  the  upper  and  the  lower 
surfaces  of  the  water  of  the  inner  beaker.  The  upper  end  of  the 
tube  being  open  it  must  extend  above  the  surface  of  the  surround- 
ing water.  Apply  gentle  heat,  stir  the  water,  and  note  the  tem- 
perature at  which  the  fat  first  begins  to  melt.  This  point  is 
indicated  by  the  initial  transparency.  For  ordinary  fats,  raise  the 
temperature  very  cautiously  from  300  C.  To  determine  the  con- 
gealing-point  remove  the  flame  and  note  the  temperature  at  which 
the  fat  begins  to  solidify.  Record  the  melting-  and  congealing- 
points  of  the  various  fats  submitted  by  the  instructor. 

Second  Method. — Fill  a  small  evaporating  dish  about  one-half 
full  of  mercury  and  place  it  on  a  water-bath.  Put  a  small  drop 
of  the  fat  under  examination  on  an  ordinary  cover  glass  and  place 
this  upon  the  surface  of  the  mercury.  Raise  the  temperature  of 
the  water-bath  slowly  and  by  means  of  a  thermometer  whose  bulb 
is  immersed  in  the  mercury,  note  the  melting-point  of  the  fat. 
Determine  the  congealing-point  by  removing  the  flame  and  leaving 
the  fat  drop  and  cover  glass  in  position  upon  the  mercury.  How 
do  the  melting-points  as  determined  by  this  method  compare  with 
those  as  determined  by  the  first  method?  Which  method  is  the 
more  accurate,  and  why? 


CHAPTER  VIII. 
PANCREATIC   DIGESTION. 

As  soon  as  the  food  mixture  leaves  the  stomach  it  comes  into 
intimate  contact  with  the  bile  and  the  pancreatic  juice.  Since  these 
fluids  are  alkaline  in  reaction  there  can  obviously  be  no  further 
peptic  activity  after  they  have  become  intimately  mixed  with  the 
chyme  and  have  neutralized  the  acidity  previously  imparted  to  it  by 
the  hydrochloric  acid  of  the  gastric  juice.  The  pancreatic  juice 
reaches  the  intestine  through  the  duct  of  Wirsung  which  opens  into 
the  intestine  near  the  pylorus. 

Normally  the  secretion  of  pancreatic  juice  is  brought  about  by  the 
stimulation  produced  by  the  acid  chyme  as  it  enters  the  duodenum. 
This  secretion  is  probably  not  due  to  a  nervous  reflex  as  was  believed 
by  Pawlow  but  rather,  as  Bayliss  and  Starling  have  shown,  is  de- 
pendent upon  the  presence,  in  the  epithelial  cells  of  the  duodenum 
and  jejunum  of  a  body  known  as  prosecretin.  This  body  is  changed 
into  secretin1  through  the  hydrolytic  action  of  the  acid  present  in  the 
chyme.  The  secretin  is  then  absorbed  by  the  blood,  passes  to  the 
pancreas  and  stimulates  the  pancreatic  cells,  causing  a  flow  of 
pancreatic  juice.  The  quantity  of  juice  secreted  under  these  con- 
ditions is  proportional  to  the  amount  of  secretin  present.  The  ac- 
tivity of  secretin  solutions  is  not  diminished  by  boiling,  hence  the 
body  does  not  react  like  an  enzyme.  Further  study  of  the  body 
may  show  it  to  be  a  definite  chemical  individual  of  relatively  low 
molecular  weight.  It  has  not  been  possible  thus  far  to  obtain  secre- 
tin from  any  tissues  except  the  mucous  membrane  of  the  duodenum 
and  jejunum. 

The  juice  as  obtained  from  a  permanent  fistula  differs  greatly 
in  its  properties  from  the  juice  as  obtained  from  a  temporary  fistula, 
and  neither  form  of  fluid  possesses  the  properties  of  the  normal 
fluid.  Pancreatic  juice  collected  by  Glaessner  from  a  natural  fistula 
has  been  found  to  be  a  colorless,  clear,  strongly  alkaline  fluid  which 
foams  readily.     It  is  further  characterized  by  containing  albumin, 

1  Secretin  belongs  to  the  class  of  substances  called  hormones  or  chemical 
messengers. 

140 


PANCREATIC    DIGESTION.  141 

globulin,  proteose  and  peptone  ;  nuclei  ipr<  >tein  isalsi  1  presenl  in  1  races. 
The  average  daily  secretion  of  pancreatic  juice  is  650  c.c.  and  it- 
specific  gravity  is  1.00S.  The  fluid  contains  1.3  per  cent  of  solid 
matter  and  the  freezing-point  is  —  0.470  C.  The  normal  pancreatic 
secretion  contains  at  least  four  distinct  enzymes.  They  are  trypsin, 
a  proteolytic  enzyme ;  pancreatic  amylase  (amyl< ipsin) ,  an  amyli >lyi ic 
enzyme;  pancreatic  lipase  (steapsin),  a  fat-splitting  enzyme:  and. 
pancreatic  renniu,  a  milk-coagulating  enzyme.  Lactase,  the  lactose- 
splitting  enzyme,  is  also  present  at  certain  times. 

The  most  important  of  the  four  enzymes  of  the  pancreatic  juice  is 
the  proteolytic  enzyme  trypsin.  This  enzyme  resembles  pepsin  in  so 
far  as  each  has  the  power  of  breaking  down  protein  material,  but 
the  trypsin  has  much  greater  digestive  power  and  is  able  to  cause  a 
more  complete  decomposition  of  the  complex  protein  molecule.  In 
the  process  of  normal  digestion  the  protein  constituents  of  the  diet 
are  for  the  most  part  transformed  into  proteoses  (albumoses)  and 
peptones  before  coming  in  contact  with  the  enzyme  trypsin.  This  is 
not  absolutely  essential  however,  since  trypsin  possesses  digestive  ac- 
tivity sufficient  to  transform  unaltered  native  proteins  and  to  produce 
from  their  complex  molecules  comparatively  simple  fragments. 
Among  the  products  of  tryptic  digestion  are  proteoses,  peptones, 
peptides,  leucine,  tyrosine,  aspartic  acid,  glutamic  acid,  alanine,  phen- 
ylalanine, glycocoll,  cystine,  serine,  valine,  proline,  oxyproline,  iso- 
leucine,  arginine,  lysine,  histidine  and  tryptophane.  (The  crystalline 
forms  of  many  of  these  products  are  reproduced  in  Chapter  IV.) 
Trypsin  does  not  occur  preformed  in  the  gland,  but  exists  there 
as  a  zymogen  called  tripsinogen  which  bears  the  same  relation  to 
trypsin  that  pepsinogen  does  to  pepsin.  Trypsin  has  never  been 
obtained  in  a  pure  form  and  therefore  very  little  can  be  stated 
definitely  as  to  its  nature.  The  enzyme  is  the  most  active  in  alkaline 
solution  but  is  also  active  in  neutral  or  slightly  acid  solutions. 
Trypsin  is  destroyed  by  mineral  acids  and  may  also  be  destroyed  by 
comparatively  weak  alkali  (2  per  cent  sodium  carbonate)  if  left  in 
contact  for  a  sufficiently  long  time.  Trypsinogen.  on  the  other 
hand,  is  more  resistant  to  the  action  of  alkalis.  In  pancreatic  di- 
gestion the  protein  does  not  swell  as  is  the  case  in  gastric  digestion, 
but  becomes  more  or  less  "  honeycombed  "  and  finally  disintegrates. 

The  pancreatic  juice  which  is  collected  by  means  of  a  fistula 
possesses  practically  no  power  to  digest  protein  matter.  A  body 
called  euterokinase  occurs  in  the  intestinal  juice  and  has  the  power 

^laessner:    Zeitschrift  fiir  physiologische  Chemie,  1904.  40.  p.  476. 


142  PHYSIOLOGICAL    CHEMISTRY. 

of  converting  tripsinogen  into  trypsin.  This  process  is  known  as 
the  "  activation  "  of  trypsinogen  and  through  it  a  juice  which  is 
incapable  of  digesting  protein  may  be  made  active.  Enterokinase 
is  not  always  present  in  the  intestinal  juice  since  it  is  secreted  only 
after  the  pancreatic  juice  reaches  the  intestine.  It  resembles  the 
enzymes  in  that  its  activity  is  destroyed  by  heat,  but  differs  mate- 
rially from  this  class  of  bodies  in  that  a  certain  quantity  is  capable 
of  activating  only  a  definite  quantity  of  trypsinogen.  It  is  how- 
ever generally  classified  as  an  enzyme.  Enterokinase  has  been  de- 
tected in  the  higher  animals,  and  a  kinase  possessing  similar  proper- 
ties has  been  shown  to  be  present  in  bacteria,  fungi,  impure  fibrin, 
lymph  glands  and  snake-venom.  The  activation  of  trypsinogen 
into  trypsin  may  be  brought  about  in  the  gland  as  well  as  in  the 
intestine  of  the  living  organism  (Mendel  and  Rettger).  The 
manner  of  the  activation  in  the  gland  and  the  nature  of  the  body 
causing  it  are  unknown  at  present. 

Delezenne  claims  that  trypsinogen  may  be  activated  by  soluble 
calcium  salts.  He  reports  experiments  which  indicate  that  pro- 
teolytically  inactive  pancreatic  juice,  obtained  directly  from  the  duct, 
when  treated  with  salts  of  this  character  assumes  the  property  of 
digesting  protein  material.  This  process  by  which  the  trypsinogen 
is  activated  through  the  instrumentality  of  calcium  salts  is  very 
rapid  and  is  designated  by  Delezenne  as  an  "  explosion."  The 
recent  suggestion  of  Mays  that  there  may  possibly  be  several  pre- 
cursors of  trypsin  one  of  which  is  activated  by  enterokinase  and 
the  others  by  other  agents,  is  of  interest  in  this  connection. 

Pancreatic  amylase  (amylopsin) ,  the  second  of  the  pancreatic  en- 
zymes, is  an  amylolytic  enzyme  which  possesses  somewhat  greater 
digestive  power  than  the  salivary  amylase  (ptyalin)  of  the  saliva. 
As  its  name  implies,  its  activity  is  confined  to  the  starches,  and 
the  products  of  its  amylolytic  action  are  dextrins  and  sugars.  The 
sugars  are  principally  iso-maltose  and  maltose  and  these  by  the 
further  action  of  an  inverting  enzyme  are  partly  transformed  into 
dextrose. 

It  is  possible  that  the  saliva  as  a  digestive  fluid  is  not  absolutely 
essential.  The  salivary  amylase  (ptyalin)  is  destroyed  by  the  hy- 
drochloric acid  of  the  gastric  juice  and  is  therefore  inactive  when 
the  chyme  reaches  the  intestine.  Should  undigested  starch  be  pres- 
ent at  this  point  however,  it  would  be  quickly  transformed  by  the 
active  pancreatic  amylase.  This  enzyme  is  not  present  in  the 
pancreatic  juice  of  infants  during  the  first  few  weeks  of  life,  thus 


PANCREATIC    DIGESTION.  143 

showing  very  clearly  that  a  starchy  diet  is  not  normal  for  this  period 
It  has  been  claimed  that  pancreatic  amylase  has  a  slight  digestive 
action  upon  unboiled  starch. 

The  third  enzyme  of  the  pancreatic  juice  is  called  pancreatic 
lipase  (steapsin)  and  is  a  fat-splitting  enzyme.  It  has  the  power 
of  splitting  the  neutral  fats  of  the  food  by  hydrolysis,  into  fatty 
acid  and  glycerol.     A  typical  reaction  would  be  as  follows : 

C3H5(a;C15H31CO)3+3H20  =  3(C15H31COOH)+C3H5(OH)3. 

Tri-palmitin.  Palmitic  acid.  Glycerol. 

Recent  researches  make  it  probable  that  fats  undergo  saponifica- 
tion to  a  certain  extent  prior  to  their  absorption.  The  fatty  acids 
formed,  in  part  unite  with  the  alkalis  of  the  pancreatic  juice  and 
intestinal  secretion  to  form  soluble  soaps ;  in  part  they  are  doubt- 
less absorbed  dissolved  in  the  bile.  Some  observers  believe  that 
the  fats  may  also  be  absorbed  in  emulsion — a  condition  promoted 
by  the  presence  of  the  soluble  soaps.  After  absorption  the  fatty 
acids  are  re-synthesized  to  form  neutral  fats  with  glycerol. 

Pancreatic  lipase  is  very  unstable  and  is  easily  rendered  inert 
by  the  action  of  acid.  For  this  reason  it  is  not  possible  to  prepare 
an  extract  having  a  satisfactory  fat-splitting  power  from  a  pancreas 
which  has  been  removed  from  the  organism  for  a  sufficiently  long 
time  to  have  become  acid  in  reaction. 

The  fourth  enzyme  of  the  pancreatic  juice  is  called  pancreatic 
rennin.  It  is  a  milk-coagulating  enzyme  whose  action  is  very 
similar  to  that  of  the  enzyme  gastric  rennin  found  in  the  gastric 
juice.  It  is  supposed  to  show  its  greatest  activity  at  a  temperature 
varying  from  6o°  to  65°  C. 


The. enzymes  of  the  intestinal  juice  are  of  great  importance  to 
the  animal  organism.  These  enzymes  include  crcpsin  (erepsase), 
sue  rase,  maltose,  lactase,  and  enterokinasc. 

Erepsin  is  a  proteolytic  enzyme  which  has  the  property  of  acting 
upon  the  proteoses  and  peptones  which  are  formed  through  the 
action  of  trypsin  and  further  splitting  them  into  amino  acids. 
Erepsin  has  no  power  of  digesting  any  native  proteins  except 
caseinogen,  histones  and  protamines.  It  possesses  its  greatest  ac- 
tivity in  an  alkaline  solution  although  it  is  slightly  active  in  acid 
solution.  An  extract  of  the  intestinal  erepsin  may  be  prepared  by 
treating  the  finely  divided  intestine  of  a  cat,  dog,  or  pig  with  toluene- 
or  chloroform-water  and  permitting  the  mixture  to  stand  with  oc- 


144  PHYSIOLOGICAL    CHEMISTRY. 

casional  shaking  for  24-72  hours.1  Enzymes  similar  to  erepsin 
occur  in  various  tissues  of  the  organism. 

The  three  invertases  sucrase,  maltase  and  lactase  are  also  im- 
portant enzymes  of  the  intestinal  mucosa.  The  sucrase  acts  upon 
sucrose  and  inverts  it  with  the  formation  of  invert  sugar  (dextrose 
and  kevulose).  Some  investigators  claim  that  sucrase  is  also 
present  in  saliva  and  gastric  juice.  It  probably  does  not  exist  nor- 
mally in  either  of  these  digestive  juices,  however,  and  if  found 
owes  its  presence  to  the  excretory  processes  of  certain  bacteria. 
Sucrases  may  also  be  obtained  from  several  vegetable  sources. 
For  investigational  purposes  it  is  ordinarily  obtained  from  yeast 
(see  p.  11).  In  exhibits  its  greatest  activity  in  the  presence  of  a 
slight  acidity  but  if  the  acidity  be  increased  to  any  extent  the  reac- 
tion is  inhibited. 

Lactase  is  an  enzyme  which  inverts  lactose  with  the  consequent 
formation  of  dextrose  and  galactose.  Its  action  is  entirely  analo- 
gous, in  type,  to  that  of  sucrase.  It  has  apparently  been  proven 
that  lactase  occurs  in  the  intestinal  mucosa  of  the  young  of  all 
animals  which  suckle  their  offspring.2  It  may  also  occur  in  the 
intestinal  mucosa  of  certain  adult  animals  if  such  animals  be  main- 
tained upon  a  ration  containing  more  or  less  lactose.  Fischer  and 
Armstrong  have   demonstrated   the   reversible   action3   of   lactase. 

For  discussions  of  maltase  and  entcrokinase  see  pages  55  and 
141  respectively. 


PREPARATION    OF    AN    ARTIFICIAL    PANCREATIC 

JUICE.4 

After  removing  the  fat  from  the  pancreas  of  a  pig  or  sheep, 
finely  divide  the  organ  by  means  of  scissors  and  grind  it  in  a  mortar. 
If  convenient,  the  use  of  an  ordinary  meat  chopper  is  a  very  satis- 
factory means  of  preparing  the  pancreas. 

When  finely  divided  as  above  the  pancreas  should  be  placed  in  a 
500  c.c.  flask,  about  150  c.c.  of  30  per  cent  alcohol  added  and  the 
flask  and  contents  shaken  frequently  for  twenty-four  hours. 
(What  is  the  reaction  of  this  alcoholic  extract  at  the  end  of  this 
period,  and  why?)      Strain  the  alcoholic  extract  through   cheese 

1  See  p.  13. 

"Mendel  and  Mitchell:  American  Journal  of  Physiology,  1907,  XX,  p.  81. 

sSee  p.  6. 

4  For  other  methods  of  preparation  see  Karl  Mays :  Zeitschrift  fur  phys- 
iologische   Chemie,  1903,  XXXVIII,  p.  428. 


PANCREATIC    DIGESTION.  H5 

cloth,   filter,  nearly  neutralize  with  potassium   hydroxide  solution 
and  then  exactly  neutralize  it  with  0.5  per  cent  sodium  carl  innate. 

Products   of   Tryptic    Digestion. 

Take  about  200  grams  of  lean  beef  which  has  been  freed  from 
fat  and  finely  ground  and  place  it  in  a  large-sized  beaker.  Intro- 
duce equal  volumes  of  the  pancreatic  extract  prepared  as  above 
and  0.5  per  cent  sodium  carbonate,  add  5  c.c.  of  an  alcoholic  solu- 
tion of  thymol  to  prevent  putrefaction,  and  place  the  beaker  in 
an  incubator  at  40 °  C.  Stir  the  contents  of  the  beaker  frequently 
and  add  more  thymol  if  it  becomes  necessary.  Allow  digestion 
to  proceed  for  from  2  to  5  days  and  then  separate  the  products 
formed  as  follows :  Strain  off  the  undissolved  residue  through 
cheese  cloth,  nearly  neutralize  the  solution  with  dilute  hydrochloric 
acid  and  then  exactly  neutralize  it  with  0.2  per  cent  hydrochloric 
acid.  A  precipitate  at  this  point  would  indicate  alkali  metaprotein 
(alkali  albuminate).  Filter  off  any  precipitate  and  divide  the  fil- 
trate into  two  parts,  a  one-fourth  and  a  three-fourth  portion. 

Transfer  the  one-fourth  portion  to  an  evaporating  dish  and 
make  the  separation  of  proteoses  and  peptones  as  well  as  the  final 
tests  upon  these  bodies  according  to  the  directions  given  on  page  114. 

Place  about  5  c.c.  of  the  three-fourth  portion  in  a  test-tube  and 
add  about  1  c.c.  of  bromine  water.  A  violet  coloration  indicates  the 
presence  of  tryptophane  (see  page  73).  Concentrate1  the  remain- 
der of  the  three-fourth  portion  to  a  thin  syrup  and  make  the  sep- 
aration of  leucine  and  tyrosine  according  to  the  directions  given  on 
page  82. 

GENERAL  EXPERIMENTS   ON   PANCREATIC 
DIGESTION. 

Experiments    on     Trypsin. 

1.  The  Most  Favorable  Reaction  for  Tryptic  Digestion. — 
Prepare  seven  tubes  as  follows : 

(a)   2-3  c.c.  of  neutral  pancreatic  extract  -f-  2-3  c.c.  of  water. 

(6)  2-3  c.c.  of  neutral  pancreatic  extract  -j-  2-3  c.c.  of  1  per 
cent  sodium  carbonate. 

(c)  2-3  c.c.  of  neutral  pancreatic  extract  -J-  2-3  c.c.  of  0.5  per 
cent  sodium  carbonate. 

1  If  the  solution  is  alkaline  in  reaction,  while  it  is  being  concentrated,  the 
amino    acids  will  be  broken  down  and  ammonia  will  be  liberated. 


I46  PHYSIOLOGICAL    CHEMISTRY. 

(d)  2-3  c.c.  of  neutral  pancreatic  extract  -f-  2-3  c.c.  of  0.2  per 
cent  hydrochloric  acid. 

(e)  2-3  c.c.  of  neutral  pancreatic  extract  +  2-3  c.c.  of  0.2  per 
cent  combined  hydrochloric  acid. 

(/)  2-3  c.c.  of  neutral  pancreatic  extract  -.]-  2-3  c.c.  of  0.4  per 
cent  boric  acid. 

(g)  2-3  c.c.  of  neutral  pancreatic  extract  -f-  2-3  c.c.  of  0.4  per 
cent  acetic  acid. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube  and 
keep  them  at  400  C.  noting  the  progress  of  digestion.  In  which 
tube  do  we  find  the  most  satisfactory  digestion,  and  why?  How 
do  the  indications  of  the  digestion  of  fibrin  by  trypsin  differ  from 
the  indications  of  the  digestion  of  fibrin  by  pepsin? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the 
following  series  of  experiments  under  tryptic  digestion  use  the 
neutral  extract  plus  an  equal  volume  of  0.5  per  cent  sodium  car- 
bonate.) In  each  of  four  tubes  place  5  c.c.  of  alkaline  pancreatic 
extract.  Immerse  one  tube  in  cold  water  from  the  faucet,  keep  a 
second  at  room  temperature  and  place  a  third  on  the  water-bath  at 
400  C.  Boil  the  contents  of  the  fourth  for  a  few  moments,  then 
cool  and  also  keep  it  at  400  C.  Into  each  tube  introduce  a  small 
piece  of  fibrin  and  note  the  progress  of  digestion.  In  which  tube 
does  the  most  rapid  digestion  occur?     What  is  the  reason? 

3.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of  tubes 
and  into  each  tube  place  6  volumes  of  water,  3  volumes  of  alkaline 
pancreatic  extract  and  1  volume  of  one  of  the  chemicals  listed  in 
Experiment  18  under  Salivary  Digestion,  page  59. 

Introduce  a  small  piece  of  fibrin  into  each  of  the  tubes  and  keep 
them  at  40 °  C.  for  one-half  hour.  Shake  the  tubes  frequently. 
In  which  tubes  do  we  get  the  least  digestion  ? 

4.  Influence  of  Bile. — Prepare  five  tubes  as  follows  : 

(a)  Five  c.c.  of  pancreatic  extract  -j-  J^-i  c.c.  of  bile. 

(b)  Five  c.c.  of  pancreatic  extract  ~f-  1-2  c.c.  of  bile. 

(c)  Five  c.c.  of  pancreatic  extract  -j-  2-3  c.c.  of  bile. 
(d.)  Five  c.c.  of  pancreatic  extract  -j-  5  c.c.  of  bile. 
(<?)  Five  c.c.  of  pancreatic  extract. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  keep  them 
at  400  C.  Shake  the  tubes  frequently  and  note  the  progress  of 
digestion.  Does  the  presence  of  bile  retard  tryptic  digestion? 
How  do  these  results  agree  with  those  obtained  under  gastric  di- 
gestion ? 


pancreatic  digestion.  1 47 

Experiments  on   Pancreatic  Amylase. 

i.  The  Most  Favorable  Reaction. — Prepare  seven  tubes  as 
follows : 

(a)  One  c.c.  of  neutral  pancreatic  extract  +  I  c.c.  of  starch  paste 
-j-  2  c.c.  of  water. 

(b)  One  c.c.  of  neutral  pancreatic  extract  -j-  I  c.c.  of  starch  paste 
+  2  c.c.  of  I  per  cent  sodium  carbonate. 

(c)  One  c.c.  of  neutral  pancreatic  extract  -|-  i  c.c.  of  starch  paste 
-\-  2  c.c.  of  0.5  per  cent  sodium  carbonate. 

(d)  One  c.c.  of  neutral  pancreatic  extract  -j-  i  c.c.  of  starch  paste 
+  2  c.c.  of  0.2  per  cent  hydrochloric  acid. 

(e)  One  c.c.  of  neutral  pancreatic  extract  -j-  i  c.c.  of  starch  paste 
-f-  2  c.c.  of  0.2  per  cent  combined  hydrochloric  acid. 

(/)  One  c.c.  of  neutral  pancreatic  extract  -j-  i  c.c.  of  starch  paste 
-j-  2  c.c.  of  0.4  per  cent  boric  acid. 

(g)  One  c.c.  of  neutral  pancreatic  extract  -f-  I  c.c.  of  starch  paste 
-f-  2  c.c.  of  0.4  per  cent  acetic  acid. 

Shake  each  tube  thoroughly  and  place  them  on  the  water-bath 
at  400  C.  At  the  end  of  a  half-hour  divide  the  contents  of  each 
tube  into  two  parts  and  test  one  part  by  the  iodine  test  and  the 
other  part  by  Fehling's  test.  Where  do  you  find  the  most  satisfac- 
tory digestion?  How  do  the  results  here  compare  with  those  ob- 
tained from  the  similar  series  under  Trypsin,  page  145. 

2.  The  Most  Favorable  Temperature. —  (For  this  and  the  fol- 
lowing series  of  experiments  upon  pancreatic  amylase  use  the 
neutral  extract  plus  an  equal  volume  of  0.5  per  cent  sodium  car- 
bonate.) In  each  of  four  tubes  place  2-3  c.c.  of  alkaline  pan- 
creatic extract.  Immerse  one  tube  in  cold  water  from  the  faucet, 
keep  a  second  at  room  temperature  and  place  a  third  on  the  water- 
bath  at  400  C.  Boil  the  contents  of  the  fourth  for  a  few  moments, 
then  cool  and  also  keep  it  at  400  C.  Into  each  tube  introduce  2-3 
c.c.  of  starch  paste  and  note  the  progress  of  digestion.  At  the  end 
of  one-half  hour  divide  the  contents  of  each  tube  into  two  parts  and 
test  one  part  by  the  iodine  test  and  the  other  part  by  Fehling's 
test.  In  which  tube  do  you  find  the  most  satisfactory  digestion  ? 
How  does  this  result  compare  with  the  result  obtained  in  the  sim- 
ilar series  of  experiments  under  Trypsin  (see  page  146)  ? 

3.  Influence  of  Metallic  Salts,  etc. — Prepare  a  series  of  tubes 
and  into  each  place  3  volumes  of  water,  3  volumes  of  alkaline  pan- 
creatic extract,  1  volume  of  one  of  the  chemicals  listed  in  Experi- 
ment   18  under   Salivary   Digestion,   page   59,   and   3   volumes   of 


I48  PHYSIOLOGICAL    CHEMISTRY. 

starch  paste.  Be  sure  to  introduce  the  starch  paste  into  the  tube  last. 
Why?  Shake  the  tubes  well  and  place  them  on  the  water-bath  at 
40  °  C.  At  the  end  of  a  half -hour  divide  the  contents  of  each  tube 
into  two  parts  and  test  one  part  by  the  iodine  test  and  the  other  part 
by  Fehling's  test.     What  are  your  conclusions  ? 

4.  Influence  of  Bile. — Prepare  five  tubes  as   follows : 

(a)  2-3  c.c.  of  pancreatic  extract  -f-  2-3  c.c.  of  starch  paste  -f- 
y2-i  c.c.  of  bile. 

(&)  2-3  c.c.  of  pancreatic  extract  -j-  2-3  c.c.  of  starch  paste  -\- 
1-2  c.c.  of  bile. 

(c)  2-3  c.c.  of  pancreatic  extract  -\-  2-3  c.c.  of  starch  paste 
-f-  2-3  c.c.  of  bile. 

(d)  2-3  c.c.  of  pancreatic  extract  +  2-3  c.c.  of  starch  paste 
-f-  .5  c.c.  of  bile. 

(e)  2-3  c.c.  of  pancreatic  extract  -{-  2-3  c.c.  of  starch  paste. 
Shake  the  tubes  thoroughly  and  place  them  on  the  water-bath 

at  400  C.  Note  the  progress  of  digestion  frequently  and  at  the 
end  of  a  half  hour  divide  the  contents  of  each  tube  into  two  parts 
and  test  one  part  by  the  iodine  test  and  the  other  part  by  Feh- 
ling's test.  What  are  your  conclusions  regarding  the  influence 
of  bile  upon  the  action  of  pancreatic  amylase? 

5.  Digestion  of  Dry  Starch. — To  a  little  dry  starch  in  a  .test- 
tube  add  about  5  c.c.  of  pancreatic  extract  and  place  the  tube  on  the 
water-bath  at  400  C.  At  the  end  of  a  half  hour  filter  and  test 
separate  portions  of  the  filtrate  by  the  iodine  and  Fettling  tests. 
What  do  you  conclude  regarding  the  action  of  pancreatic  amylase 
upon  dry  starch?  Compare  this  result  with  that  obtained  in  the 
similar  experiment  under  Salivary  Digestion  (page  58). 

6.  Digestion  of  Inulin. — To  5  c.c.  of  inulin  solution  in  a  test- 
tube  add  10  drops  of  pancreatic  extract  and  place  the  tube  on  the 
water-bath  at  400  C.  After  one-half  hour  test  the  solution  by 
Fehling's  test.1  Is  any  reducing  substance  present?  What  do 
you  conclude  regarding  the  digestion  of  inulin  by  pancreatic 
amylase  ? 

Experiments    on    Pancreatic    Lipase. 

1.  "Litmus-Milk"  Test. — Into  each  of  two  test-tubes  intro- 
duce 10  c.c.  of  milk  and  a  small  amount  of  litmus  powder.  To 
the  contents  of  one  tube  add  3  c.c.  of  neutral  pancreatic  extract 

*If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the 
pancreatic  juice,  it  will  be  necessary  to  determine  the  extent  of  the  original 
reduction  by  means  of  a  "check"  test  (see  page  47). 


PANCREATIC    DIGESTION.  149 

and  to  the  contents  of  the  other  tube  add  3  c.c.  of  water  or  ol  boiled 
neutral  pancreatic  extract.  Keep  the  tubes  at  400  C.  and  note 
any  changes  which  may  occur.  What  is  the  result  and  bow  do 
you  explain  it  ? 

2.  Ethyl  Butyrate  Test. — Into  each  of  two  test-tubes  introduce 
4  c.c.  of  water,  2  c.c.  of  ethyl  butyrate,  C;>H7COO  ■  C2H5,  and 
a  small  amount  of  litmus  powder.  To  the  contents  of  one  tube 
add  4  c.c.  of  neutral  pancreatic  extract  and  to  the  contents  of  the 
other  tube  add  4  c.c.  of  water  or  boiled  neutral  pancreatic  extract. 
Keep  the  tubes  at  400  C.  and  observe  any  changes  which  may 
occur.  What  is  the  result  and  how  do  you  explain  it?  Write  the 
equation  for  the  reaction  which  has  taken  place. 

Experiments  on  Pancreatic  Rennin. 

Prepare  four  test-tubes  as  follows : 

(a)    Five  c.c.  of  milk  -\-  10  drops  of  neutral  pancreatic  extract. 

(7?)   Five  c.c.  of  milk  -j-  20  drops  of  neutral  pancreatic  extract. 

(c)  Five  c.c.  of  milk  -j-  10  drops  of  alkaline  pancreatic  extract. 

(d)  Five  c.c.  of  milk  -\-  20  drops  of  alkaline  pancreatic  extract. 
Place  the  tubes  at  6o°-65°  C.  for  a  half  hour  without  shaking. 

Note  the  formation  of  a  clot.1  How  does  the  action  of  pancreatic 
rennin  compare  with  the  action  of  the  gastric  rennin? 

1  This  reaction  will  not  always  succeed,  owing  to  conditions  which  are 
not  well  understood. 


CHAPTER    IX 


BILE. 


The  bile  is  secreted  continuously  by  the  liver  and  passes  into  the 
intestine  through  the  common  bile  duct  which  opens  near  the 
pylorus.  Bile  is  not  secreted  continuously  into  the  intestine.  In  a 
fasting  animal  no  bile  enters  the  intestine,  but  when  food  is  taken 
the  bile  begins  to  flow ;  the  length  of  time  elapsing  between  the 
ingestion  of  the  food  and  the  secretion  of  the  bile  as  well  as  the 
qualitative  and  quantitative  characteristics  of  the  secretion  de- 
pending upon  the  nature  of  the  food  ingested.  Fats,  the  extrac- 
tives of  meat  and  the  protein  end-products  of  gastric  digestion, 
(proteoses  and  peptones)  cause  a  copious  secretion  of  bile,  whereas 
such  substances  as  water,  acids  and  boiled  starch  paste  fail  to  do 
so.  In  general  a  rich  protein  diet  is  supposed  to  increase  the 
amount  of  bile  secreted,  whereas  a  carbohydrate  diet  would  cause 
a  much  less  decided  increase  and  might  even  tend  to  decrease  the 
amount.  It  has  been  demonstrated  by  Bayliss  and  Starling  that 
the  secretion  of  bile  is  under  the  control  of  the  same  mechanism 
that  regulates  the  flow  of  pancreatic  juice  (see  p.  140).  In  other 
words,  the  hydrochloric  acid  of  the  chyme,  as  it  enters  the  duodenum 
transforms  prosecretin  into  secretin  and  this  in  turn  enters  the 
circulation,  is  carried  to  the  liver  and  stimulates  the  bile-forming 
mechanism  to  increased  activity. 

We  may  look  upon  the  bile  as  an  excretion  as  well  as  a  secretion. 
In  the  fulfillment  of  its  excretory  function  it  passes  such  bodies 
as  lecithin,  metallic  substances,  cholesterol  and  the  decomposition 
products  of  haemoglobin  into  the  intestine  and  in  this  way  aids  in  re- 
moving them  from  the  organism.  The  bile  assists  materially  in 
the  absorption  of  fats  from  the  intestine  by  its  solvent  action  on 
the  fatty  acids  formed  by  the  action  of  the  pancreatic  juice. 

The  bile  is  a  ropy,  viscid  substance  which  is  alkaline  in  reaction 
to  litmus,1  and  ordinarily  possesses  a  decidedly  bitter  taste.  It 
varies  in  color  in  the  different  animals,  the  principal  variations  being 
yellow,  brown  and  green.  Fresh  human  bile  from  the  living 
organism  ordinarily  has  a  green   or  golden-yellow   color.     Post- 

1  It  does  not  contain  any  free  hydroxyl  ions,  however. 

150 


BILE.  I  5  [ 

mortem  bile  is  variable  in  color.  It  is  very  difficult  to  determine 
accurately  the  amount  of  normal  bile  secreted  during  any  given 
period.  For  an  adult  man  it  lias  been  variously  estimated  at  from 
500  c.c.  to  1 100  c.c.  for  twenty-four  hours.  The  specific' gravity 
of  the  bile  varies  between  1.010  and  1.040,  and  the  freezing-point 
is  about  — 0.560  C.  As  secreted  by  the  liver,  the  bile  is  a  clear, 
limpid  fluid  which  contains  a  relatively  low  content  of  solid  matter. 
Such  bile  would  have  a  specific  gravity  of  approximately  1.010. 
After  it  reaches  the  gall-bladder,  however,  it  becomes  mixed  with 
mucous  material  from  the  walls  of  the  gall-bladder,  and  this  proc- 
ess coupled  with  the  continuous  absorption  of  water  from  the  bile 
has  a  tendency  to  concentrate  the  secretion.  Therefore  the  bile 
as  we  find  it  in  the  gall-bladder,  ordinarily  possesses  a  higher 
specific  gravity  than  that  of  the  freshly  secreted  fluid.  The  specific 
gravity  under  these  conditions  may  run  as  high  as  1.040. 

The  principal  constituents  of  the  bile  are  the  salts  of  the  bile  acids, 
bile  pigments,  neutral  fats,  lecithin,  pliosphatidcs  and  cholesterol, 
besides  the  salts  of  iron,  copper,  calcium  and  magnesium.  Zinc 
has  also  frequently  been  found  in  traces. 

The  bile  acids,  which  are  elaborated  exclusively  by  the  hepatic 
cells,  may  be  divided  into  two  groups,  the  glycocholic  acid  group 
and  the  taurocholic  acid  group.  In  human  bile  glycocholic  acid 
predominates,  while  taurocholic  acid  is  the  more  abundant  in  the 
bile  of  carnivora.  The  bile  acids  are  conjugate  amino-acids,  the 
glycocholic  acid  yielding  glycocoll, 

CH2NH2 
COOH, 

and  cholic  acid  upon  decomposition,  whereas  taurocholic  acid  gives 
rise  to  taurine, 

CH2NH2 

CH2S02OH, 

and  cholic  acid  under  like  conditions.  Glycocholic  acid  contains 
some  nitrogen  but  no  sulphur,  whereas  taurocholic  acid  contains  both 
these  elements.  The  sulphur  of  the  taurocholic  acid  is  present  in  the 
taurine  (amino-ethyl-sulphonic  acid),  of  which  it  is  a  characteristic 
constituent.  There  are  several  varieties  of  cholic  acid  and  there- 
fore we  have  several  forms  of  glycocholic  and  taurocholic  acids, 


152  PHYSIOLOGICAL    CHEMISTRY. 

the  variation  in  constitution  depending  upon  the  nature  of  the 
cholic  acid  which  enters  into  the  combination.  The  bile  acids  are 
present  in  the  bile  as  salts  of  one  of  the  alkalis,  generally  sodium. 
The  sodium  glycocholate  and  sodium  taurocholate  may-  be  isolated 
in  crystalline  form,  either  as  balls  or  rosettes  of  fine  needles  or  in 
the  form  of  prisms  having  ordinarily  four  or  six  sides  (Fig.  40, 
below).  The  salts  of  the  bile  acids  are  dextro-rotatory.  Among 
other  properties  these  salts  have  the  power  of  holding  the  choles- 
terol and  lecithin  of  the  bile  in  solution. 

Hammarsten  has  demonstrated  a  third  group  of  bile  acids  in 
the  bile  of  the  shark.  This  same  group  very  probably  occurs  in 
certain  other  animals  also.  These  acids  are  very  rich  in  sulphur 
and  resemble  ethereal  sulphuric  acids  inasmuch  as  upon  treatment 
with  boiling  hydrochloric  acid  they  yield  sulphuric  acid. 

Fig.  40. 


ile  Salts. 


The  bile  pigments  are  important  and  interesting  biliary  consti- 
tuents. The  following  have  been  isolated :  bilirubin,  biliverdin,  bili- 
fuscin,  biliprasin,  bilihumin,  bilicyanin,  choleprasin  and  choletelin. 
Of  these,  bilirubin  and  biliverdin  are  the  most  important  and  pre- 
dominate in  normal  bile.  The  colors  possessed  by  the  various 
varieties  of  normal  bile  are  due  almost  entirely  to  these  two  pig- 
ments, the.  biliverdin  being  the  predominant  pigment  in  greenish 
bile  and  the  bilirubin  being  the  principal  pigment  in  lighter  colored 
bile.  The  pigments,  other  than  the  two  just  mentioned,  have  been 
found  almost  exclusively  in  biliary  calculi  or  in  altered  bile  ob- 
tained at  post-mortem  examinations. 


BILE.  I  5  I 

Bilirubin,  which  is  perhaps  the  most  important  of  the  bile  pig- 
ments, is  apparently  derived  from  the  blood  pigment,  the  iron 
freed  in  the  process  being  held  in  the  liver.  Bilirubin  lias  the  same 
percentage  composition  as  ha?matoporphyrin,  which  may  be  pro- 
duced from  ■  haematin.  It  is  a  specific  product  of  the  liver  cells 
but  may  also  be  formed  in  other  parts  of  the  body.  The  pig- 
ment may  be  isolated  in  the  form  of  a  reddish-yellow  powder  or 
may  be  obtained  in  part,  in  the  form  of  reddish-yellow  rhombic 
plates   (Fig.  41,  below)   upon  the  spontaneous  evaporation  of  its 

Fig.  41. 


Bilirubin  (H^matoidin).     (Ogden.) 

chloroform  solution.  The  crystalline  form  of  bilirubin  is  prac- 
tically the  same  as  that  of  hsematoidin.  It  is  easily  soluble  in 
chloroform,  somewhat  less  soluble  in  alcohol  and  only  slightly 
soluble  in  ether  and  benzene.  Bilirubin  has  the  power  of  combin- 
ing with  certain  metals,  particularly  calcium,  to  form  combinations 
which  are  no  longer  soluble  in  the  solvents  of  the  unaltered  pig- 
ment. Upon  long  standing  in  contact  with  the  air,  the  reddish- 
yellow  bilirubin  is  oxidized  with  the  formation  of  the  green  bili- 
verdin.     Bilirubin  occurs  in  animal  fluids  as  soluble  bilirubin-alkali. 

Solutions  of  bilirubin  exhibit  no  absorption-bands.  If  an  am- 
moniacal  solution  of  bilirubin-alkali  in  water  is  treated  with  a 
solution  of  zinc  chloride,  however,  it  shows  bands  similar  to  those 
of  bilicyanin  (Absorption  Spectra,  Plate  II),  the  two  bands  be- 
tween C  and  D  being  rather  well  defined. 

Biliverdin  is  particularly  abundant  in  the  bile  of  herbivora.  It 
is  soluble  in  alcohol  and  glacial  acetic  acid  and  insoluble  in  water, 
chloroform  and  ether.  Biliverdin  is  formed  from  bilirubin  upon 
oxidation.  It  is  an  amorphous  substance,  and  in  this  (litters  from 
bilirubin  which   may  be  at   least  partly  crystallized   under  proper 


154  PHYSIOLOGICAL    CHEMISTRY. 

conditions.  Biliverdin  may  be  obtained  in  the  form  of  a  green 
powder.  In  common  with  bilirubin,  it  may  be  converted  into  hy- 
drobilirubin  by  nascent  hydrogen. 

The  neutral  solution  of  bilicyanin  or  cholecyanin  is  bluish-green 
or  steel-blue  and  possesses  a  blue  fluorescence,  the  alkaline  solution 
is  green  with  no  appreciable  fluorescence  and  the  strongly  acid  so- 
lution is  violet-blue.  The  alkaline  solution  exhibits  three  absorp- 
tion-bands, the  first  a  dark,  well-defined  band  between  C  and  D, 
somewhat  nearer  C ;  the  second  a  less  sharply-defined  band  extend- 
ing across  D  and  the  third  a  rather  faint  band  between  E  and  F, 
near  E  (Absorption  Spectra,  Plate  II).  The  strongly  acid  so- 
lution exhibits  two  absorption  bands,  both  lying  between  C  and  E 
and  separated  by  a  narrow  space  near  D.  A  third  band,  exceed- 
ingly faint,  may  ordinarily  be  seen  between  b  and  F. 

Biliary  calculi,  otherwise  designated  as  biliary  concretions  or 
gall  stones,  are  frequently  formed  in  the  gall-bladder.  These  de- 
posits may  be  divided  into  three  classes,  cholesterol  calculi,  pigment 
calculi  and  calculi  made  up  almost  entirely  of  inorganic  material. 
This  last  class  of  calculus  is  formed  principally  of  the  carbonate  and 
phosphate  of  calcium  and  is  rarely  found  in  man  although  quite 
common  to  cattle.  The  pigment  calculus  is  also  found  in  cattle, 
but  is  more  common  to  man  than  the  inorganic  calculus.  This 
pigment  calculus  ordinarily  consists  principally  of  bilirubin  in  com- 
bination with  calcium;  biliverdin  is  sometimes  found  in  small 
amount.  The  cholesterol  calculus  is  the  one  found  most  frequently 
in  man.  These  may  be  formed  almost  entirely  of  cholesterol,  in 
which  event  the  color  of  the  calculus  is  very  light,  or  they  may  con- 
tain more  or  less  pigment  and  inorganic  matter  mixed  with  the 
cholesterol,  which  tends  to  give  us  calculi  of  various  colors. 

For  discussion  of  cholesterol  see  page  250. 

Experiments    on    Bile. 

1.  Reaction. — Test  the  reaction  of  fresh  ox  bile  to  litmus. 

2.  Nucleoprotein. — Acidify  a  small  amount  of  bile  with  dilute 
acetic  acid.     A  precipitate  of  nucleoprotein  forms. 

3.  Inorganic  Constituents. — Test  for  chlorides,  sulphates  and 
phosphates  (see  page  57). 

4.  Tests  for  Bile  Pigments,  (a)  Gmelin's  Test. — To  about 
5  c.c.  of  concentrated  nitric  acid  in  a  test-tube  add  2-3  c.c.  of  diluted 
bile  carefully  so  that  the  two  fluids  do  not  mix.  At  the  point  of 
contact  note  the  various  colored  rings,  green,  blue,  violet,  red  and 


BILE.  155 

reddish-yellow.       Repeat  this  test  with  different  dilutions  of  bile 
and  observe  its  delicacy. 

(b)  Rosenbach's  Modification  of  Gmeliris  Test. — loiter  5  c.c. 
of  diluted  bile  through  a  small  filter  paper.  Introduce  a  drop  of 
concentrated  nitric  acid  into  the  cone  of  the  paper  and  note  the  suc- 
cession of  colors  as  given  in  Gmelin's  test. 

(c)  Nakaxama's  Reaction. — To  5  c.c.  of  diluted  bile  in  a  test-tube 
add  an  equal  volume  of  a  10  per  cent  solution  of  barium  chloride, 
centrifugate  the  mixture,  pour  off  the  supernatant  fluid  and  heat 
the  precipitate  with  2  c.c.  of  Nakayama's  reagent.1  In  the  presence 
of  bile  pigments  the  solution  assumes  a  blue  or  green  color. 

(d)  Huppert's  Reaction. — Thoroughly  shake  equal  volumes  of 
undiluted  bile  and  milk  of  lime  in  a  test-tube.  The  pigments  unite 
with  the  calcium  and  are  precipitated.  Filter  off  the  precipitate, 
wash  it  with  water  and  transfer  to  a  small  beaker.  Add  alcohol 
acidified  slightly  with  hydrochloric  acid  and  warm  upon  a  water- 
bath  until  the  solution  becomes  colored  an  emerald  green. 

In  examining  urine  for  bile  pigments,  according  to  Steensma,  this 
procedure  may  give  negative  results  even  in  the  presence  of  the 
pigments,  owing  to  the  fact  that  the  acid-alcohol  is  not  a  sufficiently 
strong  oxidizing  agent.  He  therefore  sug'gests  the  addition  of  a 
drop  of  a  0.5  per  cent  solution  of  sodium  nitrite  to  the  acid-alcohol 
mixture  before  warming  on  the  water-bath.  Try  this  modifica- 
tion also. 

(<?)  Hammarsten's  Reaction. — To  about  5  c.c.  of  Hammarsten's 
reagent2  in  a  small  evaporating  dish  add  a  few  drops  of  diluted  bile. 
A  green  color  is  produced.  If  more  of  the  reagent  is  now  added 
the  play  of  colors  as  observed  in  Gmelin's  test  may  be  obtained. 

(/)  Smith's  Test. — To  2-3  c.c.  of  diluted  bile  in  a  test-tube  add 
carefully  about  5  c.c.  of  dilute  tincture  of  iodine  (1  :io)  so  that  the 
fluids  do  not  mix.  A  play  of  colors,  green,  blue  and  violet,  is 
observed.  In  making  this  test  upon  the  urine  ordinarily  only  the 
green  color  is  observed. 

(g)  Salkowski-ScJiippers  Reaction. — To  10  c.c.  of  diluted  bile 
in  a  test  tube  add  5  drops  of  a  20  per  cent  solution  of  sodium 
carbonate  and  10  drops  of  a  20  per  cent  solution  of  calcium  chloride. 
Filter  off  the  resultant  precipitate  upon  a  hardened  filter-paper  and 

1  Prepared  by  combining  99  c.c.  of  alcohol  and  1  c.c.  of  fuming  hydrochloric 
acid  containing  4  grams  of  ferric  chloride  per  liter. 

2  Hammarsten's  reagent  is  made  by  mixing  1  volume  of  25  per  cent  nitric 
acid  and  19  volumes  of  25  per  cent  hydrochloric  acid  and  then  adding  1  volume 
of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol. 


I  56  PHYSIOLOGICAL    CHEMISTRY. 

> 

wash  it  with  water.  Remove  the  precipitate  to  a  small  porcelain 
dish,  add  3  c.c.  of  an  acid-alcohol  mixture1  and  a  few  drops  of  a 
dilute  solution  of  sodium  nitrite  and  heat.  The  production  of  a 
green  color  indicates  the  presence  of  bile  pigments. 

5.  Tests  for  Bile  Acids,  (a)  Pettenkofer's  Test. — To  5  c.c. 
of  diluted  bile  in  a  test-tube  add  5  drops  of  a  5  per  cent  solution 
of  sucrose.  Now  run  about  2-3  c.c.  of  concentrated  sulphuric 
acid  carefully  down  the  side  of  the  tube  and  note  the  red  ring  at  the 
point  of  contact.  Upon  slightly  agitating  the  contents  of  the  tube 
the  whole  solution  gradually  assumes  a  reddish  color.  As  the 
tube  becomes  warm,  it  should  be  cooled  in  running  water  in  order 
that  the  temperature  of  the  solution  may  not  rise  above  700  C. 

(b)  My Hits' 's  Modification  of  Pettenkofer's  Test.  To  approxi- 
mately 5  c.c.  of  diluted  bile  in  a  test-tube  add  3  drops  of  a  very 
dilute  (1  :  1,000)  aqueous  solution  of  furfurol, 

HC-CH 

II       II 
HC      C-CHO. 

\/ 

0 

Now  run  about  2—3  c.c.  of  concentrated  sulphuric  acid  carefully 
down  the  side  of  the  tube  and  note  the  red  ring  as  above.  In  this 
case  also,  upon  shaking  the  tube  the  whole  solution  is  colored  red. 
Keep  the  temperature  of  the  solution  below  yo°  C.  as  before. 

(c)  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few 
drops  of  diluted  bile  in  an  evaporating  dish  add  a  trace  of  a  dilute 
sucrose  solution  and  one  or  more  drops  of  dilute  sulphuric  acid. 
Evaporate  on  a  water-bath  and  note  the  development  of  a  violet 
color  at  the  edge  of  the  evaporating  mixture.  Discontinue  the 
evaporation  as  soon  as  the  color  is  observed. 

(d)  v.  Udrdnsky's  Test. — To  5  c.c.  of  diluted  bile  in  a  test-tube 
add  3-4  drops  of  a  very  dilute  (1  :i,ooo)  aqueous  solution  of  fur- 
furol. Place  the  thumb  over  the  top  of  the  tube  and  shake  the 
tube  until  a  thick  foam  is  formed.  By  means  of  a  small  pipette 
add  2-3  drops  of  concentrated  sulphuric  acid  to  the  foam  and  note 
the  dark  pink  coloration  produced. 

(e)  Guerin's  Reaction. — To  equal  volumes  of  diluted  bile  and 
alcohol  in  a  test-tube  add  5-6  drops  of  a  saturated  aqueous  solution 

1  Made  by  adding  5  c.c.  of  concentrated  hydrochloric  acid  to  95  c.c.  of  96  per 
cent  alcohol. 


BILK.  157 

of  furfural  and  5  6  drops  of  concentrated  sulphuric  acid.  A  blue 
color  indicates  bile  acids. 

(/)  Hay's  Test. — This  test  is  based  upon  the  principle  thai  bile- 
acids  have  the  property  of  reducing  the  surface  tension  of  fluids 
in  which  they  are  contained.  The  test  is  performed  as  follows : 
Cool  about  10  c.c.  of  diluted  bile  in  a  test-tube  to  17°  C.  or  lower 
and  sprinkle  a  little  finely  pulverized  sulphur  upon  the  surface  of 
the  fluid.  The  presence  of  bile  acids  is  indicated  if  the  sulphur 
sinks  to  the  bottom  of  the  liquid,  the  rapidity  with  which  the  sulphur 
sinks  depending  upon  the  quantity  of  bile  acids  present  in  the  mix- 
ture. The  test  is  said  to  react  with  bile  acids  when  they  are  present 
in  the  proportion  1  :  120,000. 

Some  investigators  claim  that  it  is  impossible  to  differentiate  be- 
tween bile  acids  and  bile  pigments  by  this  test. 

6.  Crystallization  of  Bile  Salts. — To  25  c.c.  of  undiluted  bile 
in  an  evaporating  dish  add  enough  animal  charcoal  to  form  a 
paste  and  evaporate  to  dryness  on  a  water-bath.  Remove  the  resi- 
due, grind  it  in  a  mortar  and  transfer  it  to  a  small  flask.  Add 
about  50  c.c.  of  95  per  cent  alcohol  and  boil  on  a  water-bath  for 
20  minutes.  Filter,  and  add  ether  to  the  filtrate  until  there  is  a 
slight  permanent  cloudiness.  Cover  the  vessel  and  stand  it  away 
until  crystallization  is  complete.  Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  shown  in  Fig.  40,  page  152. 
Try  one  of  the  tests  for  bile  acids  upon  some  of  the  crystals. 


i58 


PHYSIOLOGICAL    CHEMISTRY. 


7.  Analysis  of  Biliary  Calculi. — Grind  the  calculus  in  a  mortar 
with  10  c.c.  of  ether.     Filter. 


Filtrate   I. 


Allow  to  evaporate  and  examine 
for  cholesterol  crystals  (Fig.  42, 
p.  159).  (For  further  tests  see  ex- 
periment 8,  below.) 


Residue   I. 

(On  paper  and  in  mortar.) 

I 

Treat  with  dilute  hydrochloric 
acid  and  filter. 


Filtrate   II. 

Test  for  calcium,  phos- 
phates and  iron.  Evapo- 
rate remainder  of  filtrate 
to  dryness  in  porcelain 
crucible  and  ignite.  Dis- 
solve residue  in  dilute 
hydrochloric  acid  and 
make  alkaline  with  am- 
monium hydroxide.  Blue 
color    indicates    copper. 


Residue   II. 

(On  paper  and  in  mortar.) 
Wash   with   a   little   water.     Dry  the   filter 
paper. 

Treat  with  5  c.c.  chloroform  and  filter. 


Filtrate  III.  Residue  III. 

Bilirubin.  (On   paper   and   in 

(Apply  test  for  bile       mortar.) 
pigments.) 

Treat  with  5  c.c.  of 
hot  alcohol. 


Biliverdin. 

S.  Tests  for  Cholesterol. 

(a)  Microscopical  Examination. — Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  shown  in  Fig.  42,  page 

159- 

(b)  Iodine-Sulphuric  Acid  Test. — Place  a  few  crystals  of  choles- 
terol in  one  of  the  depressions  of  a  test-tablet  and  treat  with  a  drop 
of  concentrated  sulphuric  acid  and  a  drop  of  a  very  dilute  solution 
of  iodine.  A  play  of  colors  consisting  of  violet,  blue,  green  and  red 
results. 

(c)  The  Liebermann-Bur  chard  Test. — Dissolve  a  few  crystals  of 
cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  sulphuric 
acid.  The  solution  becomes  red,  then  blue,  and  finally  bluish- 
green  in  color. 

(d)  Salkou'ski's  Test. — Dissolve  a  few  crystals  of  cholesterol  in 
a  little  chloroform  and  add  an  equal  volume  of  concentrated  sul- 
phuric acid.     A  play  of  colors  from  bluish-red  to  cherry-red  and 


i:ilk. 


'59 


purple  is  noted  in  the  chloroform  while  the  acid  assumes  a  marked 
green  fluorescence. 

(e)   Schiff's  Reaction.-— To  a  little  cholesterol  in  an  evaporating 
dish  add  a  few  drops  of  Schiff's  reagent.'1      Evaporate  to  dryness 


Cholesterol. 


over   a  low   flame   and   observe   the   reddish-violet   residue   which 
changes  to  a  bluish-violet. 

9.  Preparation  of  Taurine. — To  300  c.c.  of  bile  in  a  casserole 
add  100  c.c.  of  hydrochloric  acid  and  heat  until  a  sticky  mass 
(dyslysin)  is  formed.  This  point  may  be  determined  by  drawing- 
out  a  thread-like  portion  of  the  mass  by  means  of  a  glass  rod,  and 
if  it  solidifies  immediately  and  assumes  a  brittle  character  we  may 
conclude  that  all  the  taurocholic  and  glycochblic  acid  has  been 
decomposed.  Decant  the  solution  and  concentrate  it  to  a  small  vol- 
ume on  the  water-bath.  Filter  the  hot  solution  to  remove  sodium 
chloride  and  other  substances  which  may  have  separated,  and 
evaporate  the  filtrate  to  dryness.  Dissolve  the  residue  in  5  per 
cent  hydrochloric  acid  and  precipitate  with  ten  volumes  of  95  per 
cent  alcohol.  Filter  off  the  taurine  and  recrystallize  it  from  hot 
water.  (Save  the  alcoholic  filtrate  for  the  preparation  of  glycocoll, 
page  160.)     Alake  the  following  tests  upon  the  taurine  crystals: 

(a)  Examine   them   under   the   microscope   and   compare    with 

Fig.  43,  P-  l6°- 

(b)  Heat  a  crystal  upon  platinum   foil.     The   taurine  at  first 

1  Schiff's  reagent  consists  of  a  mixture  of  three  volumes  of  concentrated 
sulphuric  acid  and  one  volume  of  10  per  cent  ferric  chloride. 


i6o 


PHYSIOLOGICAL    CHEMISTRY. 


melts,  then  turns  brown  and  finally  carbonizes  as  the  temperature  is 
raised.     Note  the  suffocating  odor.     What  is  it? 

(c)  Test  the  solubility  of  the  crystals  in  water  and  in  alcohol. 

(d)  Grind  up  a  crystal  with  four  times  its  volume  of  dry  sodium 
carbonate  and  fuse  on  platinum  foil.  Cool  the  residue,  transfer 
it  to  a  test-tube  and  dissolve  it  in  water.  Add  a  little  dilute  sul- 
phuric acid  and  note  the  odor  of  hydrogen  sulphide.     Hold  a  piece 


Fig.  43. 


Taurine. 


of  filter  paper,  moistened  with  a  small  amount  of  lead  acetate,  over 
the  opening  of  the  test-tube  and  observe  the  formation  of  lead  sul- 
phide. 

10.  Preparation  of  Glycocoll. — Concentrate  the  alcoholic  filtrate 
from  the  last  experiment  (9)  until  no  more  alcohol  remains.  The 
glycocoll  is  present  here  in  the  form  of  an  hydrochloride  and  may 
be  liberated  from  this  combination  by  the  addition  of  freshly  pre- 
cipitated lead  hydroxide  or  by  lead  hydroxide  solution.  Remove 
the  lead  by  hydrogen  sulphide.  Filter  and  decolorize  the  filtrate 
by  animal  charcoal.  Filter  again,  concentrate  the  nitrate  and  set  it 
aside  for  crystallization.  Glycocoll  separates  as  colorless  crystals 
(Fig.  44,  p.  161), 

n.  Synthesis  of  Hippuric  Acid. — To  some  of  the  glycocoll  pre- 
pared in  the  last  experiment  or  furnished  by  the  instructor,  add 
a  little  water,  about  1  c.c.  of  benzoyl  chloride  and  render  alkaline 
with  potassium  hydroxide  solution.  Stopper  the  tube  and  shake 
it  until  no  more  heat  is  evolved.  Now  render  strongly  alkaline 
with  potassium  hydroxide  and  shake  the  mixture  until  no  odor  of 


MILK. 


161 


benzoyl  chloride  can  be  detected.  Cool,  acidify  with  hydrochloric 
acid,  add  an  equal  volume  of  petroleum  ether  and  shake  thoroughly 
to  remove  the  benzoic  acid.      (Evaporate  this   solution  and  note 


Fig.  44. 


Glycocoll. 

the  crystals  of  benzoic  acid.  Compare  them  with  those  shown 
in  Fig.  94,  page  289.)  Decant  the  ethereal  solution  into  a  porcelain 
dish  and  extract  again  with  ether.  The  hippuric  acid  remains  in 
the  aqueous  solution.  Filter  it  off  and  wash  it  with  a  small  amount 
of  cold  water  while  still  on  the  filter.  Remove  it  to  a  small,  shallow 
vessel,  dissolve  it  in  a  small  amount  of  hot  water  and  set  it  aside  for 
crystallization.  Examine  the  crystals  microscopically  and  com- 
pare them  with  those  in  Fig.  92,  page  282. 

The  chemistry  of  the  synthesis  is  represented  thus : 


CH2NH2  COC1  OCNHCH.COOH. 

+  11=11  +  HO. 


COOH 

Glycocoll.  Benzoyl  chloride.  Hippuric  acid. 


CHAPTER   X. 

PUTREFACTION  PRODUCTS. 

The  putrefactive  processes  in  the  intestine  are  the  result  of  the 
action  of  bacteria  upon  the  protein  material  present.  This  bac- 
terial action  which  is  the  combined  effort  of  many  forms  of  micro- 
organisms is  confined  almost  exclusively  to  the  large  intestine. 
Some  of  the  products  of  the  putrefaction  of  proteins  are  identical 
with  those  formed  in  tryptic  digestion,  although  the  decomposition 
of  the  protein  material  is  much  more  extensive  when  subjected 
to  putrefaction.  Some  of  the  more  important  of  the  putrefaction 
products  are  the  following :  Indole,  skatole,  paracresol,  phenol,  para- 
oxyphenylpropionic  acid,  para-oxyphenylacetic  acid,  volatile  fatty 
acids,  hydrogen  sulphide,  methane,  methyl  mercaptan,  hydrogen, 
and  carbon  dioxide,  beside  proteoses,  peptones,  ammonia  and  amino 
acids.  Of  these  the  indole,  skatole,  phenol  and  paracresol  appear 
in  part  in  the  urine  as  ethereal  sulphuric  acids,  whereas  the  oxyacids 
mentioned  pass  unchanged  into  the  urine.  The  potassium  indoxyl 
sulphate  (page  279)  content  of  the  urine  is  a  rough  indicator  of  the 
extent  of  the  putrefaction  within  the  intestine. 

The  portion  of  the  indole  which  is  excreted  in  the  urine  is  first 
subjected  to  a  series  of  changes  within  the  organism  and  is  sub- 
sequently eliminated  as  indican.  These  changes  may  be  represented 
thus : 

/\ CH  /\ C(OH) 

I       I      II      +0  =  |      J      || 
WCH  \/\/CH 

NH  NH 

Indole.  Indoxyl. 

/\ C(OH)  /\ C(0-S03H) 

I       I      ||  +  H2S04  =  I       I      ||  +H20 

\/\/CH  \/\/CH 
NH  NH 

Indoxyl.  Indoxyl  sulphuric  acid. 

In  the  presence  of  potassium  salts  the  indoxyl  sulphuric  acid  is 
then  transformed  into  indoxyl  potassium  sulphate  (or  indican), 

162 


PUTREFACTION    PRODUCTS.  1 63 

/\ C(0-SO,K), 

\/\/CH 
NH 

and  eliminated  as  such  in  the  urine. 

Indican  may  be  decomposed  by  treatment  with  concentrated  hy- 
drochloric acid  (see  tests  on  page  280)  into  sulphuric  acid  and  in- 
doxyl.  The  latter  body  may  then  be  oxidized  to  form  indigo-blue 
thus : 

/\ C(OH)  /\ CO  OC /\ 

2  1       I      ||  +20  =  1       II  III  +2H20 

\A/CH  \/\/C=C\/\/ 

NH  NH  NH 

Tndoxyl.  Indigo-blue. 

This  same  reaction  may  also  occur  under  pathological  conditions 
unthin  the  organism,  thus  giving  rise  to  the  appearance  of  crystals 
of  indigo-blue  in  the  urine. 

Skatole  is  likewise  changed  within  the  organism  and  eliminated 
in  the  form  of  a  chromogenic  substance.  Skatole  is,  however,  of 
less  importance  as  a  putrefaction  product  than  indole  and  ordinarily 
occurs  in  much  smaller  amount.  The  tryptophane  group  of  the 
protein  molecule  yields  the  indole  and  skatole  formed  in  intestinal 
putrefaction,  but  the  reasons  for  the  transformation  of  the  major 
portion  of  this  tryptophane  into  indole  and  the  minor  portion  into 
skatole  are  not  well  understood.     Indole  is  more  toxic  than  skatole. 

Phenol  occurs  in  fairly  large  amount  in  certain  abnormal  con- 
ditions of  the  organism,  but  ordinarily  the  amount  is  very  small. 
It  is  probably  derived  from  the  tyrosine  group  of  the  protein  mole- 
cule. Phenol  is  conjugated  in  the  liver  to  form  phenyl  potassium 
sulphate  and  appears  in  the  urine  in  this  form  (Baumann  and 
Herter).  Para-cresol  occurs  in  the  urine  as  cresyl  potassium  sul- 
phate. 

Regarding  the  claim  of  Nencki  that  methyl  mercaptan  is  formed 
as  a  g'as  during  intestinal  putrefaction  it  is  an  important  fact  that 
Herter1  has  been  unable  to  detect  the  mercaptan  in  fresh  feces.  He 
is  therefore,  not  inclined  to  accept  the  theory  that  methyl  mercap- 
tan is  formed  in  ordinary  intestinal  putrefaction  but  believes  that 
it  may  be  formed  in  exceptional  cases.  Hydrogen  sulphide  is.  how- 
ever, formed  in  all  cases  of  intestinal  putrefaction. 

1  Herter :    Bacterial  Infections  of  the  Digestive  Tract,  p.  22*. 


164  physiological  chemistry. 

Experiments   on    Putrefaction    Products. 

In  many  courses  in  physiological  chemistry  the  instructors  are  so 
limited  for  time  that  no  extended  study  of  the  products  of  putre- 
faction can  very  well  be  attempted.  Under  such  conditions  the 
scheme  here  submitted  may  be  used  profitably  in  the  way  of  a  dem- 
onstration. Where  the  number  of  students  is  not  too  great,  a 
single  large  putrefaction  may  be  started,  and,  after  the  initial 
distillation,  both  the  resulting  distillate  and  residue  may  be  dis- 
tributed to  the  members  of  the  class  for  individual  manipulation. 

Preparation  of  Putrefaction  Mixture. — Place  a  weighed  mix- 
ture of  coagulated  egg  albumin  and  ground  lean  meat  in  a  flask  or 
bottle  and  add  approximately  2  liters  of  water  for  every  kilogram 
of  protein  used.  Sterilize  the  vessel  and  contents,  inoculate  with 
the  colon  bacillus  and  keep  at  40 °  C.  for  two  or  three  weeks.  If 
cultures  of  the  colon  bacillus  are  not  available,  add  60  c.c.  of  a  cold 
saturated  solution  of  sodium  carbonate  for  every  liter  of  water  pre- 
viously added  and  inoculate  with  some  putrescent  material  (pan- 
creas or  feces).1  Mix  the  putrefaction  mixture  very  thoroughly 
by  shaking  and  insert  a  cork  furnished  with  a  glass  tube  to  which 
is  attached  a  wash  bottle  containing  a  3  per  cent  solution  of  mer- 
curic cyanide.2  This  device  is  for  the  purpose  of  collecting  the 
methyl  mercaptan,  a  gas  formed  during  the  process  of  putrefac- 
tion. It  also  serves  to  diminish  the  odor  arising  from  the  putre- 
fying material.  Place  the  putrefaction  mixture  at  400  C.  for  two 
or  three  weeks  and  at  the  end  of  that  time  make  a  separation  of 
the  products  of  putrefaction  according  to  the  following  directions : 

Subject  the  mixture  to  distillation  until  the  distillate  and  residue 
are  approximately  equal  in  volume. 

1  Putrefying  protein  may  be  prepared  by  treating  10  grams  of  finely  ground 
lean  meat  with  100  c.c.  of  water  and  2  c.c.  of  a  saturated  solution  of  sodium 
carbonate  and  keeping  the  mixture  at  400  C.   for  twenty-four  hours. 

-  Concentrated  sulphuric  acid  containing  a  small  amount  of  isatin  may  be 
used  as  a  substitute  for  mercuric  cyanide.  When  this  modification  is  employed 
it  is  necessary  to  use  calcium  chloride  tubes  to  exclude  moisture  from  the 
isatin  solution. 


PUTREFACTION    PRODUCTS. 


I65 


PART   I 


MANIPULATION    OF    THE    DISTILLATE. 

Acidify  with  hydrochloric  acid  and  extract  with  ether. 


Ether  Extract  No.  1. 
Add  an  equal  volume  of  water, 
make  alkaline  with  potassium  hy- 
droxide  and   shake   thoroughly. 


Residue  No.  1. 

Allow  the  ether  to  volatilize. 
Evaporate  and  detect  ammonium 
chloride  crystals  (Fig.  45,  p.  166). 


Ether  Extract  No.  2. 
Evaporate  spontaneously.  Indole 
and  skatole   remain.     Try  proper 
reactions  (see  pages  168  and  170). 


Alkaline  Solution  No.  1. 
Acidify  with  hydrochloric  acid, 
add  sodium  carbonate  and  extract 
with  ether. 


Ether  Extract  No.  3. 
Evaporate.     Detect  phenol  and 
cresol    (paracresol).     See    p.    170. 


Alkaline  Solution  No.  2. 
Acidify  with  hydrochloric  acid, 
and   extract   with   ether. 


Ether  Extract  No.  4. 
Evaporate.     Volatile  fatty  acids 
remain. 


Final  Residue. 
(Discard.) 


DETAILED    DIRECTIONS    FOR    MAKING    THE 

SEPARATIONS    INDICATED    IN 

THE   SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be  conven- 
iently conducted  in  a  separatory  funnel.  Mix  the  fluids  for  ex- 
traction in  the  ratio  of  lzv,o  volumes  of  ether  to  three  volumes  of  the 
distillate.  Shake  very  thoroughly  for  a  few  moments  then  draw 
off  the  extracted  fluid  and  add  a  new  portion  of  the  distillate.  Re- 
peat the  process  until  the  entire  distillate  has  been  extracted.  Add 
a  small  amount  of  fresh  ether  at  each  extraction  to  replace  that 
dissolved  by  the  water  in  the  preceding-  extraction. 

Residue  No.  1. — Unite  the  portions  of  the  distillate  extracted  as 
above  and  allow  the  ether  to  volatilize  spontaneously.  Evaporate 
until  crystallization  begins.  Examine  the  crystals  under  the  micro- 
scope.    Ammonium  chloride  predominates.     Explain  its  presence. 


1 66  PHYSIOLOGICAL    CHEMISTRY. 

Ether  Extract  No.  I. — Add  an  equal  volume  of  water,  render  the 
mixture  alkaline  with  potassium  hydroxide  and  shake  thoroughly 
by  means  of  a  separatory  funnel  as  before.     The  volatile  fatty  acids, 

Fig.  45. 


Ammonium  Chloride. 

contained  among  the  putrefaction  products,  would  be  dissolved  by 
the  alkaline  solution  (No.  1)  whereas  any  indole  or  skatole  would 
remain  in  the  ethereal  solution  (No.  2). 

Alkaline  Solution  No.  1. — Acidify  with  hydrochloric  acid  and  add 
sodium  carbonate  solution  until  the  fluid  is  neutral  or  slightly  acid 
from  the  presence  of  carbonic  acid.  At  this  point  a  portion 
of  the  solution,  after  being  heated  for  a  few  moments,  should  pos- 
sess an  alkaline  reaction  on  cooling.  Extract  the  whole  mixture 
with  ether  in  the  usual  way,  using  care  in  the  manipulation  of  the 
stop  cock  to  relieve  the  pressure  due  to  the  evolution  of  carbon 
dioxide.  The  ether  (Ether  Extract  No.  3)  removes  any  phenol 
or  cresol  which  may  be  present  while  the  volatile  fatty  acids  will 
remain  in  the  alkaline  solution  (No.  2)  as  alkali  salts. 

Ether  Extract  No.  2. — Drive  off  the  major  portion  of  the  ether 
at  a  low  temperature  on  a  water-bath  and  allow  the  residue  to  evap- 
orate spontaneously.  Indole  and  skatole  should  be  present  here. 
Prove  the  presence  of  these  bodies.  For  tests  for  indole  and 
skatole  see  pp.  168  and  170. 

Alkaline  Solution  No.  2. — Make  strongly  acid  with  hydrochloric 
acid  and  extract  with  a  small  amount  of  ether,  using  a  separatory 
funnel.  As  carbon  dioxide  is  liberated  here,  care  must  be  used  in 
the  manipulation  of  the  stop  cock  of  the  funnel  in  relieving  the 


PUTREFACTION    PRODUCTS. 


l6/ 


pressure  within  the  vessel.  The  volatile  fatty  acids  are  dissolved 
by  the  ether  (Ether  Extract  No.  4). 

Ether  Extract  No.  J. — Evaporate  this  ethereal  solution  on  a 
water-bath.  The  oily  residue  contains  phenol  and  cresol.  The 
cresol  is  present  for  the  most  part  as  paracresol.  Add  some  water 
to  the  oily  residue  and  heat  it  in  a  flask.  Cool  and  prove  the  pres- 
ence of  phenol  and  cresol.     For  tests  for  these  bodies  see  page  170. 

Ether  Extract  No.  4. — Evaporate  on  a  water-bath.  The  volatile 
fatty  acids  remain  in  the  residue. 


PART    II. 
MANIPULATION   OF  THE  RESIDUE. 

Evaporate,  filter  and  extract  with  ether. 


Ether  Extract. 
Evaporate,    extract   the   residue 
with  warm  water  and  filter. 


Aqueous  Solution. 
Evaporate  until  crystals  hegin  to 
from.     Stand  in  a  cold  place  until 
crystallization  is  complete.    Filter. 


Crystalline  Deposit. 
Consists  of  a  mixture 
of  leucine  and  tyrosine 
crystals.  (Figs.  23,  26 
and  104,  pages  72,  76  and 
350.) 


Filtrate  No.  1. 
Contains  proteose,  pep- 
tone, aromatic  acids  and 
tryptophane. 


Filtrate  No.  2. 
Contains  oxyacids  and 
skatolc-carbonic   acid. 


Residue. 
Contains     non-volatile 
fatty  acids. 


DETAILED  DIRECTIONS  FOR  MAKING  THE 
SEPARATIONS   INDICATED   IN 
THE    SCHEME. 

Preliminary  Ether  Extraction. — This  extraction  may  be  con- 
ducted in  a  separatory  funnel.  In  order  to  make  a  satisfactory  ex- 
traction the  mixture  should  be  shaken  very  thoroughly.  Separate 
the  ethereal  solution  from  the  aqueous  portion  and  treat  them  ac- 
cording to  the  directions  given  on  p.  168. 


1 68  PHYSIOLOGICAL    CHEMISTRY. 

Ether  Extract. — Evaporate  this  solution  on  a  safety  water-bath 
until  the  ether  has  been  entirely  removed.  Extract  the  residue  with 
warm  water  and  filter. 

Aqueous  Solution. — Evaporate  this  solution  until  crystallization 
begins.  Stand  the  solution  in  a  cold  place  until  no  more  crystals 
form.  This  crystalline  mass  consists  of  impure  leucine  and  tyro- 
sine.    Filter  off  the  crystals. 

Crystalline  Deposit. — Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  reproduced  in  Figs.  23,  26  and  104, 
pages  J2,  76  and  350.  Do  the  forms  of  the  crystals  of  leucine  and 
tyrosine  resemble  those  previously  examined?  Make  a  separation 
of  the  leucine  and  tyrosine  and  apply  typical  tests  according  to 
directions  given  on  pages  83  and  84. 

Filtrate  No.  1. — Make  a  test  for  tryptophane  with  bromine  water 
(see  page  145).,  and,  also  with  the  Hopkins-Cole  reagent  (see  page 
91).  Use  the  remainder  of  the  filtrate  for  the  separation  of  pro- 
teoses and  peptones.  Make  the  separation  according  to  the  direc- 
tions given  on  page  114. 

Filtrate  No.  2. — This  solution  contains  para-oxyphenylacetic  acid, 
para-oxyphenylpropionic  acid  and  skatole-carbonic  acid.  Prove  the 
presence  of  these  bodies  by  appropriate  tests.  Tests  for  oxyacids 
and  skatole-carbonic  acid  are  given  on  page  171. 


TESTS  FOR  VARIOUS  PUTREFACTION   PRODUCTS. 

Tests    for    Indole. 

1.  Herter's  /3-Naphthaquinone  Reaction. — (a)  To  a  dilute 
aqueous  solution  of  indole  (1:500,000)  add  one  drop  of  a  2  per 
cent  solution  of  /?-naphthaquinone-sodium-monosulphonate.  No 
reaction  occurs.  Add  a  drop  of  a  10  per  cent  solution  of  potassium 
hydroxide  and  note  the  gradual  development  of  a  blue  or  blue-green 
color  which  fades  to  green  if  an  excess  of  the  alkali  is  added.  Ren- 
der the  green  or  blue-green  solution  acid  and  note  the  appearance  of 
a  pink  color.  Heat  facilitates  the  development  of  the  color  re- 
action. 

One  part  of  indole  in  one  million  parts  of  water  may  be  detected 
by  means  of  this  test  if  carefully  performed. 

(b)  If  the  alkali  be  added  to  a  more  concentrated  indole  solu- 
tion before  the  introduction  of  the  naphthaquinone  the  course  of 
the  reaction  is  different,  particularly  if  the  indole  solution  is  some- 


PUTREFACTION  PRODUCTS.  169 

what  more  concentrated  than  that  mentioned  above  and  if  heal  is 
used.  Under  these  conditions  the  blue  indole  compound  ultimately 
forms  as  fine  acicnlar  crystals  which  rise  to  the  surface. 

If  we  do  not  wait  for  the  production  of  the  crystalline  body  but  as 
soon  as  the  blue  color  forms,  shake  the  aqueous  solution  with  chlor- 
oform, the  blue  color  disappears  from  the  solution  and  the  chloro- 
form assumes  a  pinkish-red  hue.  This  is  a  distinguishing  feature 
of  the  indole  reaction  and  facilitates  the  differentiation  of  indole 
from  other  bodies  which  yield  a  similar  blue  color. 

2.  Konto's  Reaction. — Distil  the  solution  to  be  tested  until  only 
one-third  of  the  original  solution  remains.  Make  the  distillate  al- 
kaline with  sodium  hydroxide  and  distil  again  in  order  to  separate 
the  indole  from  the  phenol,  the  latter  remaining  in  the  residue.  In- 
asmuch as  this  second  distillate  generally  contains  a  large  amount 
of  ammonia  it  should  be  acidified  with  dilute  sulphuric  acid  and 
again  distilled.  To  i  c.c.  of  this  ammonia-free  distillate  in  a  test- 
tube  add  3  drops  of  a  40  per  cent  solution  of  formaldehyde  and 
1  c.c.  of  concentrated  sulphuric  acid.  Now  agitate  the  mixture 
and  note  the  appearance  of  a  violet  red  color  if  a  trace  of  indole 
is  present.  The  test  is  said  to  serve  for  the  detection  of  indole 
when  present  in  a  dilution  of  1  1700,000. 

Skatole  gives  a  yellow  or  brown  color  under  the  above  conditions. 

3.  Cholera-red  Reaction. — To  a  little  of  the  residue  in  a  test- 
tube  add  one-tenth  its  volume  of  a  0.02  per  cent  solution  of  potas- 
sium nitrite  and  mix  thoroughly.  Carefully  run  concentrated  sul- 
phuric acid  down  the  side  of  the  tube  so  that  it  forms  a  layer  at 
the  bottom.  Note  the  purple  color.  Neutralize  with  potassium 
hydroxide  and  observe  the  production  of  a  bluish-green  color. 

4.  Legal's  Reaction. — To  a  small  amount  of  the  residue  in  a 
test-tube  add  a  few  drops  of  a  freshly  prepared  solution  of  sodium 
nitroprusside,  Na2Fe(CN)5NO  -j-  2H0O.  Render  alkaline  with 
potassium  hydroxide  and  note  the  production  of  a  violet  color.  If 
the  solution  is  now  acidified  with  glacial  acetic  acid  the  violet  is 
transformed  into  a  blue. 

5.  Pine  Wood  Test.  — Moisten  a  pine  splinter  with  concentrated 
hydrochloric  acid  and  insert  it  into  the  residue.  The  wood  as- 
sumes a  cherry-red  color. 

6.  Nitroso-indole  Nitrate  Test. — Acidify  some  of  the  residue 
with  nitric  acid,  add. a  few  drops  of  a  potassium  nitrite  solution  and 
note  the  production  of  a  red  precipitate  of  nitroso-indole  nitrate.  If 
the  residue  contains  but  little  indole  simply  a  red  coloration  will 


170  PHYSIOLOGICAL    CHEMISTRY. 

result.  Compare  this  result  with  the  result  of  the  similar  test  on 
skatole. 

Tests  for  Skatole. 

i.  Herter's    Para-dimethylaminobenzaldehyde    Reaction.1 — 

To  5  c.c.  of  the  distillate  or  aqueous  solution  under  examination 
add  i  c.c.  of  an  acid  solution  of  para-dimethylaminobenzaldehyde2 
and  heat  the  mixture  to  boiling.  A  purplish-blue  coloration  is 
produced3  which  may  be  intensified  through  the  addition  of  a  few 
drops  of  concentrated  hydrochloric  acid.  If  the  solution  be  cooled 
under  running  water  it  loses  its  purplish  tinge  of  color  and  becomes 
a  definite  blue.  The  solution  at  this  point  may  be  somewhat  opal- 
escent through  the  separation  of  uncombined  para-dimethylamino- 
benzaldehyde. Care  should  be  taken  not  to  add  an  excess  of  hy- 
drochloric acid  inasmuch  as  the  end-reaction  has  a  tendency  to  fade 
under  the  influence  of  a  high  acidity. 

A  rough  idea  regarding  the  actual  quantity  of  skatole  in  a  mix- 
ture may  be  obtained  by  extracting  this  blue  solution  with  chloro- 
form and  subsequently  comparing  this  chloroform  solution,  by 
means  of  a  colorimeter  (Duboscq),  with  the  maximal  reaction,  ob- 
tained with  a  skatole  solution  of  known  strength. 

2.  Color  Reaction  with  Hydrochloric  Acid. — Acidify  some  of 
the  residue  with  concentrated  hydrochloric  acid.  Note  the  pro- 
duction of  a  violet  color. 

3.  Acidify  some  of  the  residue  with  nitric  acid  and  add  a  few 
drops  of  a  potassium  nitrite  solution.  Note  the  white  turbidity. 
Compare  this  result  with  the  result  of  the  similar  test  on  indole. 

Tests  for  Phenol  and  Cresol. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  re- 
agent. A  red  color  results.  Compare  this  test  with  the  similar  one 
under  Tyrosine  (  see  page  83 ) . 

2.  Ferric  Chloride  Test. — Add  a  few  drops  of  neutral  ferric 
chloride  solution  to  a  little  of  the  residual  fluid.  A  dirty  bluish- 
gray  color  is  formed. 

3.  Formation  of  Bromine  Compounds. — Add  some  bromine 
water  to  a  little  of  the  fluid  under  examination.  Note  the  crys- 
talline precipitate  of   tribromphenol  and  tribromcresol. 

1Herter:    Bacterial  Infections  of  the  Digestive  Tract,  1907,  p.  141. 

2  Made  by  dissolving  5  grams  of  para-dimethylaminobenzaldehyde  in  100  c.c. 
of  10  per  cent  sulphuric  acid. 

3  If  the  color  does  not  appear  add  more  of  the  aldehyde  solution. 


PUTREFACTION    PRODUCTS.  I  7  I 

Tests  for  Oxyacids. 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  re- 
agent.    A  red  color  results. 

2.  Bromine  Water  Test. — Add  a  few  drops  of  bromine  water 
to  some  of  the  filtrate.     A  turbidity  or  precipitate  is  observed. 

Test  for  Skatole-carbonic  Acid. 

Ferric  Chloride  Test. — Acidify  some  of  the  filtrate  with  hydro- 
chloric acid,  add  a  few  drops  of  ferric  chloride  solution  and  heat. 
Compare  the  end-reaction  with  that  given  by  phenol. 


CHAPTER   XI 


FECES. 


The  feces  is  the  residual  mass  of  material  remaining  in  the  intes- 
tine after  the  full  and  complete  exercise  of  the  digestive  and  ab- 
sorptive functions  and  is  ultimately  expelled  from  the  body  through 
the  rectum.  The  amount  of  this  fecal  discharge  varies  with  the 
individual  and  the  diet.  Upon  an  ordinary  mixed  diet  the  daily  ex- 
cretion by  an  adult  male  will  aggregate  1 10-170  grams  with  a  solid 
content  ranging  between  25  and  45  grams;  the  fecal  discharge  of 


Fig.  46. 


Microscopical  Constituents  of  Feces,     (v.  Jaksch.) 

a,  Muscle  fibers  ;  b,  connective  tissue  ;  c,  epithelium  ;  d,  leucocytes  ;  e,  spiral  cells  ; 
f,  g,  h,  h  various  vegetable  cells  ;  k,  "  triple  phosphate  "  crystals  ;  /,  woody  vegetable 
cells ;  the  whole  interspersed  with  innumerable  micro-organisms  of  various  kinds. 


such  an  individual  upon  a  vegetable  diet  will  be  much  greater  and 
may  even  be  as  great  as  350  grams  and  possess  a  solid  content  of 
75  grams.  The  variation  in  the  normal  daily  output  being  so  great 
renders  this  factor  of  very  little  value  for  diagnostic  purposes, 
except  where  the  composition  of  the  diet  is  accurately  known. 
Lesions  of  the  digestive  tract,  a  defective  absorptive  function  or 
increased  peristalsis  as  well  as  an  admixture  of  mucus,  pus,  blood 
and  pathological  products  of  the  intestinal  wall  may  cause  the  total 
amount  of  excrement  to  be  markedly  increased. 

172 


FECES. 


1/3 


The  fecal  pigment  of  the  normal  adult  is  hydrobilirubin.  This 
pigment  originates  from  the  bilirubin  which  is  secreted  into  the  in- 
testine in  the  bile,  the  transformation  from  bilirubin  to  hydrobili- 
rubin being  brought  about  through  the  activity  of  certain  bacteria. 
Hydrobilirubin  is  sometimes  called  stercobilin  and  bears  a  close  re- 
semblance to  urobilin  or  may  even  be  identical  with  that  pigment. 
Neither  bilirubin  nor  biliverdin  occurs  normally  in  the  fecal  dis- 
charge of  adults,  although  the  former  may  be  detected  in  the  ex- 
crement of  nursing  infants.  The  most  important  factor,  however, 
in  determining  the  color  of  the  fecal  discharge  is  the  diet.  A 
mixed  diet  for  instance  produces 
stools  which  vary  in  color  from 
light  to  dark  brown,  an  exclusive 
meat  diet  gives  rise  to  a  brown- 
ish-black stool,  whereas  the  stool 


resulting"  from  a  milk  diet  is  in- 
variably light  colored.  Certain 
pigmented  foods  such  as  the  chlo- 
rophyllic  vegetables,  and  various 
varieties  of  berries,  each  afford 
stools  having  a  characteristic 
color.  Certain  drugs  act  in  a 
similar  way  to  color  the  fecal  dis- 
charge. This  is  well  illustrated  by 
the  occurrence  of  green  stools  following  the  use  of  calomel  and  of 


H/EMATOiDix    Crystals   from   Acholic 
Stools,      {v.  Jaksch.) 

Color  of  crystals  same  as  the  color  of 
those  in  Fig.  41,  p.   153. 


black  stools  after  bismuth  ingestion. 


The  green  color  of  the  calo- 


mel stool  is  generally  believed  to  be  due  to  biliverdin.  v.  Jaksch. 
however,  claims  to  have  proven  this  view  to  be  incorrect  since  he 
was  able  to  detect  hydrobilirubin  (or  urobilin)  but  no  biliverdin 
in  stools  after  the  administration  of  calomel.  The  bismuth  stool 
derives  its  color  from  the  black  sulphide  which  is  formed  from 
the  subnitrate  of  bismuth.  In  cases  of  biliary  obstruction  the 
grayish-white  acholic  stool  is  formed. 

Under  normal  conditions  the  odor  of  feces  is  due  to  skatole  and 
indole,  two  bodies  formed  in  the  course  of  putrefactive  processes 
occurring  within  the  intestine  (see  page  162).  Such  todies  as 
methane,  methyl  mercaptan  and  hydrogen  sulphide  may  also  add 
to  the  disagreeable  character  of  the  odor.  The  intensity  of  the 
odor  depends  to  a  large  degree  upon  the  character  of  the  diet,  being 
very  marked  in  stools  from  a  meat  diet,  much  less  marked  in 
stools   from  a  vegetable  diet  and   frequently  hardly  detectable  in 


174  PHYSIOLOGICAL    CHEMISTRY. 

stools  from  a  milk  diet.  Thus  the  stool  of  the  infant  is  ordi- 
narily nearly  odorless  and  any  decided  odor  may  generally  be  read- 
ily traced  to  some  pathological  source. 

A  neutral  reaction  ordinarily  predominates  in  normal  stools  al- 
though slightly  alkaline  or  even  acid  stools  are  met  with.     The 
acid  reaction  is  encountered  much  less  frequently 
FlG-  48-  than  the  alkaline  and  then  commonly  only  fol- 

lowing a  vegetable  diet. 

The  form  and  consistency  of  the  stool  is  de- 
pendent, in  large  measure,  upon  the  nature  of 
the  diet  and  particularly  upon  the  quantity  of 
water  ingested.  Under  normal  conditions  the 
consistency   may   vary    from   a  thin,   pasty   dis- 

Charcot-Leyden  io,ii-i 

Crystals.  charge  to  a  firmly  formed  stool,     btools  which 

are  exceedingly  thin  and  watery  ordinarily  have  a 
pathological  significance.  In  general  the  feces  of  the  carnivorous 
animals  is  of  a  firmer  consistency  than  that  of  the  herbivora. 

It  is  frequently  desirable  for  clinical  or  experimental  purposes 
to  make  an  examination  of  the  fecal  output  which  constitutes  the 
residual  mass  from  a  certain  definite  diet.  Under  such  conditions, 
it  is  customary  to  cause  the  person  under  observation  to  ingest 
some  substance,  at  the  beginning  and  end  of  the  period  in  ques- 
tion, which  shall  sufficiently  differ  in  color  and  consistency  from  the 
surrounding  feces  as  to  render  comparatively  easy  the  differentiation 
of  the  feces  of  that  period  from  the  feces  of  the  immediately  pre- 
ceding and  succeeding  periods.  One  of  the  most  satisfactory  meth- 
ods of  making  this  "  separation  "  is  by  means  of  the  ingestion 
of  a  gelatin  capsule  containing  about  0.2  gram  of  powdered  char- 
coal at  the  beginning  and  end  of  the  period  under  observation. 
This  procedure  causes  the  appearance  of  tzt'.o  black  zones  of  char- 
coal in  the  fecal  mass  and  thus  renders  comparatively  simple,  the 
differentiation  of  the  feces  of  the  intermediate  period.  Some 
similar  method  for  the  "  separation  of  feces  "  is'  universally  prac- 
ticed in  connection  with  the  scientifically  accurate  type  of  nutri- 
tion or  metabolism  experiment  which  embraces  the  collection  of 
useful  data  regarding  the  income  and  outgo  of  nitrogen,  and  other 
elements. 

Among  the  macroscopical  constituents  of  the  feces  may  be  men- 
tioned the  following :  Intestinal  parasites,  undigested  food  particles, 
gall  stones,  pathological  products  of  the  intestinal  wall,  enteroliths, 
intestinal  sand  and  objects  which  have  been  accidentally  swallowed. 


FECES.  175 

The  fecal  constituents  which  at  various  times  and  under  different 
conditions  may  be  detected  by  the  use  of  the  microscope  are  as  fol- 
lows:  Constituents  derived  from  the  food,  such  as  muscle  fibers, 
connective  tissue  shreds,  starch  granules  and  fat;  formed  elements 
derived  from  the  intestinal  tract,  such  as  epithelium,  erythrocytes 
and  leucocytes;  mucus;  pus  corpuscles;  parasites  and  bacteria.  In 
addition  to  the  constituents  named,  the  following  crystalline  deposits 
may  be  detected:  cholesterol,  soaps,  fatty  acid,  fat,  bismuth  sul- 
phide, liccmatoidin.  "  triple  phosphate,"  C hare ot-Ley den  crystals 
and  the  oxalate,  carbonate,  phosphate,  sulphate  and  lactate  of  cal- 
cium. 

The  detection  of  minute  quantities  of  blood  in  the  feces  ("  oc- 
cult blood")  has  recently  become  a  recognized  aid  to  a  correct 
diagnosis  of  certain  disorders.  In  these  instances  the  hemorrhage 
is  ordinarily  so  slight  that  the  identification  by  means  of  macro- 
scopical  characteristics  as  well  as  the  microscopical  identification 
through  the  detection  of  erythrocytes  are  both  unsatisfactory  in 
their  results.  Of  the  tests  given  for  the  detection  of  "  occult 
blood"  the  aloin-turpentinc  test  (page  178)  is  probably  the  most 
satisfactory.  Since  "  occult  blood "  occurs  with  considerable 
regularity  and  frequency  in  gastrointestinal  cancer  and  in  gastric 
and  duodenal  ulcer,  its  detection  in  the  feces  is  of  especial  value  as 
an  aid  to  a  correct  diagnosis  of  these  disorders. 

It  has  been  quite  clearly  shown  that  the  intestine  of  the  newly 
born  is  sterile.  However  this  condition  is  quickly  altered  and  bac- 
teria may  be  present  in  the  feces  before  or  after  the  first  inges- 
tion of  food.  There  are  three  possible  means  of  infecting  the 
intestine,  i.  e.,  by  way  of  the  mouth  or  anus  or  through  the  blood. 
The  infection  by  means  of  the  blood  seldom  occurs  except  under 
pathological  conditions,  thus  limiting  the  general  infection  to  the 
mouth  and  anus. 

In  infants  with  pronounced  constipation  two-thirds  of  the  dry 
substance  of  the  stools  has  been  found  to  consist  of  bacteria.  In 
the  stools  of  normal  adults  probably  about  one-third  of  the  dry 
substance  is  bacteria.1  The  average  excretion  of  dry  bacteria  in 
twenty-four  hours  for  an  adult  is  about  eight  grams. 

Some  of  the  more  important  organisms  met  with  in  the  feces  are 
the  following  :2  B.  coli,  B.  lactis  aerogenes,  Bact.  JJ'elchii,  B. 
bifidus  and  coccal  forms.     Of  these  the  first  three  types  mentioned 

1  Schittenhelm  and  Tollens  found  bacteria  to  comprise  42  per  cent  of  the  dry 
matter.     This  value  is,  however,  probably  too  high. 
'  Herter  and  Kendall:    Journal  of  Biological  Chemistry,  1908,  V,  p.  283. 


176  PHYSIOLOGICAL    CHEMISTRY. 

are  gas- forming  organisms.  The  production  of  gas  by  the  fecal 
flora  in  dextrose-bouillon  is  subject  to  great  variations  under  path- 
ological conditions  :  alterations  in  the  diet  of  normal  persons  will 
also  cause  wide  fluctuations.  In  this  connection  Herter  has  ob- 
served a  marked  reduction  or  even  complete  cessation  of  gas  pro- 
duction by  the  mixed  fecal  bacteria  while  considerable  doses  of 
benzoate  were  being  given.  A  return  to  the  former  plane  of  gas 
production  followed  .the  discontinuation  of  the  benzoate.1  Data 
as  to  the  production  of  gas  are  of  considerable  importance  in  a  diag- 
nostic way  although  the  exact  cause  of  the  variations  is  not  yet  es- 
tablished. It  should  be  borne  in  mind  in  this  connection  that  gas 
volumes  are  frequently  variable  with  the  same  individual.  For 
this  reason  it  is  necessary  in  every  instance  to  follow  the  gas  pro- 
duction for  a  considerable  period  of  time  before  drawing  conclu- 
sions.2 

For  diagnostic  purposes  the  macroscopical  and  microscopical  ex- 
aminations of  the  feces  ordinarily  yield  much  more  satisfactory  data 
than  are  secured  from  its  chemical  examination. 

Experiments    on    Feces. 

1.  Macroscopical  Examination. — If  the  stool  is  watery  pour 
it  into  a  shallow  dish  and  examine  directly.  If  it  is  firm  or  pasty 
it  should  be  treated  with  water  and  carefully  stirred  before  the 
examination  for  macroscopical  constituents  is  attempted. 

The  macroscopical  constituents  may  be  collected  very  satisfactor- 
ily by  means  of  a  Boas  sieve  (Fig.  49,  page  177).  This  sieve  is 
constructed  of  two  easily  detachable  hemispheres  which  are  held 
together  by  means  of  a  bayonet  catch.  In  using  the  apparatus  the 
feces  is  spread  out  upon  a  very  fine  sieve  contained  in  the  lower 
hemisphere  and  a  stream  of  water  is  allowed  to  play  upon  it 
through  the  medium  of  an  opening  in  the  upper  hemisphere.  The 
apparatus  is  provided  with  an  orifice  in  the  upper  hemisphere 
through  which  the  feces  may  be  stirred  by  means  of  a  glass  rod 
during  the  washing  process.  After  15-30  minutes  washing  noth- 
ing but  the  coarse  fecal  constituents  remain  upon  the  sieve. 

2.  Microscopical  Examination. — Watery  stools  should  be  placed 
in  a  shallow  dish,  thoroughly  mixed  and  a  small  amount  removed 
to  a  slide  for  examination.  Stools  of  a  firm  or  pasty  consistency 
should  be  rubbed  up  in  a  mortar  with  physiological  salt  solution 

1  Private  communication  from  Professor  C.  A.  Herter. 

2  Herter  and  Kendall :  he.  cit. 


FECES. 


177 


Fig.   49. 


Boas'  Sieve. 


and  a  small  portion  of  the  resulting-  mixture  transferred  to  a  slide 
for  examination.  In  normal  feces  look  for  food  particles,  bacteria 
and  crystalline  bodies.  In  pathological  stools,  in 
addition  to  these  substances,  look  for  animal 
parasites  and  pathological  products  of  the  intes- 
tinal wall.     See  Fig.  46,  page  172. 

3.  Reaction. — Thoroughly  mix  the  feces  and 
apply  moist  red  and  blue  litmus  papers  to  the  sur- 
face. If  the  stool  is  hard  it  should  be  mixed  with 
water  before  the  reaction  is  taken.  Examine  the 
stool  as  soon  after  defecation  as  is  convenient, 
since  the  reaction  may  change  very  rapidly.  The 
reaction  of  the  normal  stools  of  adult  man  is 
ordinarily  neutral  or  faintly  alkaline  to  litmus, 
but  seldom  acid.  Infants'  stools  are  generally 
acid  in  reaction. 

4.  Starch. — If  any  imperfectly  cooked  starch- 
containing'  food  has  been  ingested  it  will  be  pos- 
sible to  detect  starch  granules  by  a  microscopical  examination  of 
the  feces.  If  the  granules  are  not  detected  by  a  microscopical  ex- 
amination, the  feces  should  be  placed  in  an  evaporating  dish  or 
casserole  and  boiled  with  water  for  a  few  minutes.  Filter  and  test 
the  filtrate  by  the  iodine  test  in  the  usual  way  (see  page  44). 

5.  Cholesterol  and  Fat. — Extract  the  dry  feces  with  ether  in 
a  Soxhlet  apparatus  (see  Fig.  125).  If  this  apparatus  is  not  avail- 
able transfer  the  dry  feces  to  a  flask,  add  ether  and  shake  fre- 
quently for  a  few  hours.  Filter  and  remove  the  ether  by  evapora- 
tion. The  residue  contains  cholesterol  and  the  mixed  fats  of  the 
feces.  For  every  gram  of  fat  add  about  i}4  gram  of  solid 
potassium  hydroxide  and  25  c.c.  of  95  per  cent  alcohol  and  boil  in 
a  flask  on  a  water-bath  for  one-half  hour,  maintaining  the  volume 
of  alcohol  constant.  This  alcoholic-potash  has  saponified  the  mixed 
fats  and  we  now  have  a  mixture  of  soaps  and  cholesterol.  Add 
sodium  chloride,  in  substance,  to  the  mixture  and  extract  with 
ether  to  dissolve  out  the  cholesterol.  Remove  the  ether  by  evapora- 
tion and  examine  the  residue  microscopically  for  cholesterol  crystals. 
Try  any  of  the  other  tests  for  cholesterol  as  given  on  page  158. 

6.  Blood. — Undecomposed  blood  may  be  detected  macroscopi- 
cally.  If  uncertain,  look  for  erythrocytes  under  the  microscope, 
and  spectroscopically  for  the  spectrum  of  oxyhemoglobin  (see 
Absorption  Spectra,  Plate  I). 


13 


178  PHYSIOLOGICAL    CHEMISTRY. 

In  case  the  blood  has  been  altered  or  is  present  in  minute  amount 
("occult  blood"),  and  cannot  be  detected  by  the  means  just  men- 
tioned, the  following  tests  may  be  tried  : 

(a)  Aloin-Turp entitle  Test. — Mix  the  stool  very  thoroughly  and 
take  about  5  grams  of  the  mixture  for  the  test.  Reduce  this  sam- 
ple to  a  semi-fluid  mass  by  means  of  distilled  water  and  extract 
very  thoroughly  with  an  equal  volume  of  ether  to  remove  any  fat 
which  may  be  present.  Now  treat  the  extracted  feces  with  one- 
third  its  volume  of  glacial  acetic  acid  and  10  c.c.  of  ether  and  ex- 
tract very  thoroughly  as  before.  The  acid-ether  extract  will  rise 
to  the  top  and  may  be  removed. 

Introduce  2-3  c.c.  of  this  acid-ether  solution  into  a  test-tube, 
add  an  equal  volume  of  a  dilute  solution  of  aloin  in  70  per  cent  al- 
cohol and  2-3  c.c.  of  ozonized  turpentine  and  shake  the  tube  gently. 
If  blood  is  present  the  entire  volume  of  fluid  ordinarily  becomes 
pink  and  finally  cherry  red.  In  some  instances  the  color  will  be 
limited  to  the  aloin  solution  which  sinks  to  the  bottom.  This  color 
reaction  should  occur  within  fifteen  minutes  in  order  to  indicate  a 
positive  test  for  blood,  since  the  aloin  will  turn  red  of  itself  if 
allowed  to  stand  for  a  longer  period.  The  color  is  ordinarily 
light  yellow  in  a  negative  test.  Hydrogen  peroxide  is  not  a  satis- 
factory substitute  for  turpentine  in  this  test. 

(b)  Weber's  Guaiac  Test. — Mix  a  little  feces  with  30  per  cent 
acetic  acid  to  form  a  fluid  mass.  Transfer  to  a  test-tube  and  ex- 
tract with  ether.  If  blood  is  present  the  ether  will  assume  a  brown- 
ish-red color.  Filter  off  the  ether  extract  and  to  a  portion  of  the 
filtrate,  add  an  alcoholic  solution  of  guaiac  (strength  about  1  :  60), 1 
drop  by  drop,  until  the  fluid  becomes  turbid.  Now  add  hydrogen 
peroxide  or  old  turpentine.  In  the  presence  of  blood  a  blue  color 
is  produced  (see  page  196). 

(c)  Cozme's  Guaiac  Test. — To  1  gram  of  moist  feces  add  4-5  c.c. 
of  glacial  acetic  acid  and  extract  the  mixture  with  30  c.c.  of  ether. 
To  1-2  c.c.  of  the  extract  add  an  equal  volume  of  water,  agitate 
the  mixture,  introduce  a  few  granules  of  powdered  guaiac  resin, 
and  after  bringing  the  resin  into  solution,  gradually  add  30  drops 
of  old  turpentine  or  hydrogen  peroxide.  A  blue  color  indicates 
the  presence  of  blood.  Cowie  claims  that  by  means  of  this  test 
an  intestinal  hemorrhage  of  1  gram  can  easily  be  detected  by  an 
examination  of  the  feces. 

1  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid 
instead  of  an  alcoholic  solution  of  guaiac  resin. 


FECES.  179 

(d)  Acid-Hcematin. — Examine  some  of  the  ethereal  extract  from 
Experiment  ( b )  spectroscopically.  Note  the  typical  spectrum  of 
acid-hsematin   (see  Absorption  Spectra,   I  Mate  II). 

7.  Hydrobilirubin.  Schmidt's  Test. — Rub  up  a  small  amount  of 
feces  in  a  mortar  with  a  concentrated  aqueous  solution  of  mercuric 
chloride.  Transfer  to  a  shallow,  flat-bottomed  dish  and  allow  to 
stand  6-24  hours.  The  presence  of  hydrobilirubin  will  be  indicated 
by  a  deep  red  color  being;  imparted  to  the  particles  of  feces  con- 
taining this  pigment.  1  "his  red  color  is  due  to  the  formation  of 
hydrobilirubin-mercury.  If  unaltered  bilirubin  is  present  in  any 
portion  of  the  feces  that  portion  will  be  green  in  color  due  to  the 
oxidation  of  bilirubin  to  biliverdin. 

Another  method  for  the  detection  of  hydrobilirubin  is  the  fol- 
lowing: Treat  the  dry  feces  with  absolute  alcohol  acidified  with 
sulphuric  acid  and  shake  thoroughly.  The  acidified  alcohol  extracts 
the  pigment  and  assumes  a  reddish  color.  Examine  a  little  of 
this  fluid  spectroscopically  and  note  the  typical  spectrum  of  hydro- 
bilirubin (Absorption  Spectra,  Plate  II). 

8.  Bilirubin.1  (a)  Gmelin's  Test. — Place  a  few  drops  of  con- 
centrated nitric  acid  in  an  evaporating  dish  or  on  a  porcelain  test- 
tablet  and  allow  a  few  drops  of  feces  and  water  to  mix  with  it. 
The  usual  play  of  colors  of  Gmelin's  test  is  produced,  i.  e.,  green, 
blue,  violet,  red  and  yellow.  If  so  desired,  this  test  may  be  exe- 
cuted on  a  slide  and  observed  under  the  microscope. 

(b)  Happert's  Test. — Treat  the  feces  with  water  to  form  a  semi- 
fluid mass,  add  an  equal  amount  of  milk  of  lime,  shake  thorough- 
ly and  filter.  Wash  the  precipitate  with  water,  then  transfer  both 
the  paper  and  the  precipitate  to  a  small  beaker  or  flask,  add  a  small 
amount  of  95  per  cent  alcohol  acidified  slightly  with  sulphuric  acid 
and  heat  to  boiling  on  a  water-bath.  The  presence  of  bilirubin  is 
indicated  by  the  alcohol  assuming  a  green  color. 

Steensma  advises  the  addition  of  a  drop  of  a  0.5  per  cent  solu- 
tion of  sodium  nitrite  to  the  acid-alcohol  mixture  before  warming 
on  the  water-bath.     Try  this  modification  also. 

9.  Bile  Acids. — Extract  a  small  amount  of  feces  with  alcohol 
and  filter.  Evaporate  the  filtrate  on  a  water-bath  to  drive  off  the 
alcohol  and  dissolve  the  residue  in  water  made  slightly  alkaline  with 
potassium  hydroxide.  Upon  this  aqueous  solution  try  any  of  the 
tests  for  bile  acids  given  on  page  156. 

1The  detection  of  bilirubin  in  the  feces  is  comparatively  simple  provided  it 
is  not  accompanied  by  other  pigments.  When  other  pigments  are  present,  how- 
ever, it  is  difficult  to  detect  the  bilirubin  and  at  times,  ma}-  be  found  impossible. 


1  8o  PHYSIOLOGICAL    CHEMISTRY. 

10.  Caseinogen. — Extract  the  fresh  feces  first  with  a  dilute  so- 
lution of  sodium  chloride,  and  later  with  water  acidified  with  dilute 
acetic  acid,  to  remove  soluble  proteins.  Now  extract  the  feces  with 
0.5  per  cent  sodium  carbonate  and  filter.  Add  dilute  acetic  acid 
to  the  filtrate  to  precipitate  the  caseinogen,  being  careful  not  to  add 
an  excess  of  the  reagent  as  the  caseinogen  would  dissolve.  Filter 
off  the  caseinogen  and  test  it  according  to  directions  given  on  page 
224.  Caseinogen  is  found  principally  in  the  feces  of  children  who 
have  been  fed  a  milk  diet.-  Mucin  would  also  be  extracted  by  the 
dilute  alkali,  if  present  in  the  feces.  What  test  could  you  make  on 
the  newly  precipitated  body  to  differentiate  between  mucin  and 
caseinogen  ? 

11.  Nucleoprotein. — Mix  the  stool  thoroughly  with  water,  trans- 
fer to  a  flask,  and  add  an  equal  amount  of  saturated  lime  water. 
Shake  frequently  for  a  few  hours,  filter,  and  precipitate  the  nucleo- 
protein with  acetic  acid.  Filter  off  this  precipitate  and  test  it  as 
follows : 

(a)  Phosphorus. — Test  for  phosphorus  by  fusion  (see page  251). 

(b)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 

(c)  Protein  Color  Test. — Try  any  of  the  protein  color  tests. 
What   proof   have   you   that  the   above   body   was  not   mucin? 

What  other  test  can  you  use  to  differentiate  between  nucleoprotein 
and  mucin  ? 

12.  Albumin  and  Globulin. — Extract  the  fresh  feces  with  a 
dilute  solution  of  sodium  chloride.  (The  preliminary  extract  from 
the  preparation  of  caseinogen  (10),  above,  may  be  utilized  here.) 
Filter,  and  saturate  a  portion  of  the  filtrate  with  sodium  chloride 
in  substance.  A  precipitate  signifies  globulin.  Filter  off  the  pre- 
cipitate and  acidify  the  filtrate  slightly  with  dilute  acetic  acid.  A 
precipitate  at  this  point  signifies  albumin.  Make  a  protein  color 
test  on  each  of  these  bodies. 

13.  Proteose  and  Peptone. — Heat  to  boiling  the  portion  of  the 
sodium  chloride  extract  not  used  in  the  last  experiment.  Filter  off 
the  coagulum,  if  any  forms.  Acidify  the  filtrate  slightly  with 
acetic  acid  and  saturate  with  sodium  chloride  in  substance.  A  pre- 
cipitate here  indicates  proteose.  Filter  it  off  and  test  it  according 
to  directions  given  on  page  115.  Test  the  filtrate  for  peptone  by 
the  biuret  test. 

14.  Inorganic  Constituents. — Prepare  a  dilute  aqueous  solution 
of  dry  feces  and  decolorize  it  by  means  of  purified  animal  charcoal. 
Make  the  following  tests  upon  the  clear  solution : 


FECES.  I  S  I 

(a)  Chlorides. — Acidify  with  nitric  acid  and  add  argentic 
nitrate. 

(b)  Phosphates. — Acidify  with  nitric  acid,  add  molybdic  solution 
and  warm  gently. 

(c)  Sulphates. — Acidify  with  hydrochloric  acid,  add  barium 
chloride  and  warm. 

15.  Konto's  Reaction  for  Indole. — Rub  up  the  stool  with  water 
to  form  a  thin  paste.  From  this  point  the  test  is  the  same  as  for  the 
detection  of  indole  in  putrefaction  mixtures  (see  page  169). 

16.  Schmidt's  Nuclei  Test. — This  test  serves  as  an  aid  to  the 
diagnosis  of  pancreatic  insufficiency.  The  test  is  founded  upon 
the  theory  that  cell  nuclei  are  digestible  only  in  pancreatic  juice, 
and  therefore  that  the  appearance  in  the  feces  of  such  nuclei  indi- 
cates insufficiency  of  pancreatic  secretion.  The  procedure  is  as 
follows :  Cubes  of  fresh  beef  about  one-half  centimeter  square 
are  enclosed  in  small  gauze  bags  and  ingested  with  a  test  meal. 
Subsequently  the  fecal  mass  resulting  from  this  test-meal  is  exam- 
ined, the  bag  opened  and  the  condition  of  the  enclosed  residue  de- 
termined. Under  normal  conditions  the  nuclei  would  be  digested. 
Therefore  if  the  nuclei  are  found  to  be  for  the  most  part  undi- 
gested, and  the  intervening  period  has  been  sufficient  to  permit  of  the 
full  activity  of  the  pancreatic  function  (at  least  6  hours),  it  may 
be  considered  a  sign  of  pancreatic  insufficiency. 

It  has  been  claimed  by  Steele  that  under  certain  conditions  the 
non-digestion  of  the  nuclei  may  indicate  a  general  lowering  of  the 
digestive  power  rather  than  a  true  pancreatic  insufficiency. 


CHAPTER    XII 


BLOOD. 


Blood  is  composed  of  four  types  of  form-elements  (erythrocytes 
or  red  blood  corpuscles,  leucocytes  or  white  blood  corpuscles,  blood 
plates  or  plaques  and  blood  dust  or  hsemoconien)  held  in  suspen- 
sion in  a  fluid  called  blood  plasma.  These  form-elements  compose 
about  60  per  cent  of  the  blood,  by  weight.  Ordinarily  blood  is  a 
dark  red,  opaque  fluid  due  to  the  presence  of  the  red  blood  corpus- 
cles, but  through  the  action  of  certain  substances  such  as  water, 
ether  or  chloroform  it  may  be  rendered  transparent.  Blood  so 
altered  is  said  to  be  laked.  The  laking  process  is  simply  a  liberation 
of  the  haemoglobin  from  the  stroma  of  the  red  blood  corpuscle. 
Normal  blood  is  alkaline  in  reaction1  to  litmus,  the  alkalinity  being 
due  principally  to  sodium  carbonate  and  phosphate.  The  specific 
gravity  of  the  blood  of  adults  ordinarily  varies  between  1.045  and 
1.075.  It  varies  somewhat  with  the  sex,  the  blood  of  males  hav- 
ing a  rather  higher  specific  gravity  than  that  of  females  of  the 
same  species.  Under  pathological  conditions  also  the  density  of 
the  blood  may  be  very  greatly  altered.  The  freezing'-point  (A)  of 
normal  blood  is  about  — 0.560  C.  Variations  between  — 0.5 1° 
and  0.62 °  C.  may  be  due  entirely  to  dietary  conditions,  but  if  any 
marked  variation  is  noted  it  can  in  most  cases  be  traced  to  a  dis- 
ordered kidney  function.  The  total  amount  of  blood  in  the  body 
has  been  variously  estimated  at  from  one-twelfth  to  one-fourteenth 
of  the  body  weight.     Perhaps  1/13.5  is  the  most  satisfactory  figure. 

Among  the  most  important  constituents  of  blood  plasma  are  the 
four  protein  bodies,  fibrinogen,  nucleoprotein,  serum  globulin  (eu- 
globulin  and  pseudo-globulin)  and  scrum  albumin.  Plasma  con- 
tains about  8.2  per  cent  of  solids  of  which  the  protein  constituents 
named  above  constitute  approximately  84  per  cent  and  the  inor- 
ganic constituents  (mainly  chlorides,  phosphates  and  carbonates) 
approximately  10  per  cent.  Among  the  inorganic  'constituents 
sodium  chloride  predominates.  To  prevent  coagulation,  blood 
plasma  is  ordinarily  studied  in  the  form  of  an  oxalated  or  salted 

1  Recently  it  has  been  shown  by  physico-chemical  methods  that  the  blood 
is  in  reality  neutral  in  reaction. 

182 


BLOOD.  183 

plasma.     The  former  may  be  obtained  by  allowing  the  blood   to 

flow  from  an  opened  artery  into  an  equal  volume  of  0.2  per  cent 
ammonium  oxalate  solution,  whereas  in  the  preparation  of  a  salted 
plasma  10  per  cent  sodium  chloride  solution  may  be  used  as  the 
diluting  fluid. 

Fibrinogen  is  perhaps  the  most  important  of  the  protein  con- 
stituents of  the  plasma.  It  is  also  found  in  lymph  and  chyle  as 
well  as  in  certain  exudates  and  transudates.  Fibrinogen  possesses 
the  general  properties  of  the  globulins,  but  differs  from  serum  globu- 
lin in  being  precipitated  upon  half -saturation  with  sodium  chloride. 
In  the  process  of  coagulation  of  the  blood  the  fibrinogen  is  trans- 
formed into  fibrin.  This  fibrin  is  one  of  the  principal  constituents 
of  the  ordinary  blood-clot. 

The  nucleoprotein  of  blood  possesses  many  of  the  characteristics 
of  serum  globulin.  In  common  with  this  body  it  is  easily  soluble 
in  sodium  chloride,  and  is  completely  precipitated  from  its  solu- 
tions upon  saturation  with  magnesium  sulphate.  It  is  much  less 
soluble  in  dilute  acetic  acid  than  serum  globulin  and  its  solutions 
coagulate  at  6$°-6g°  C. 

The  body  formerly  called  serum  globulin  is  probably  not  an  in- 
dividual substance.  Recent  investigations  seem  to  indicate  that  it 
may  be  resolved  into  two  individual  bodies  called  eu  globulin  and 
pseudo  globulin.  The  euglobulin  is  practically  insoluble  in  water  and 
may  be  precipitated  in  the  presence  of  28-36  per  cent  of  saturated 
ammonium  sulphate  solution.  The  pseudoglobulin,  on  the  contrary, 
is  soluble  in  water  and  is  only  precipitated  by  ammonium  sulphate 
in  the  presence  of  from  36  to  44  per  cent  of  saturated  ammonium 
sulphate  solution. 

In  common  with  serum  globulin  the  body  known  as  serum  albu- 
min seems  also  to  consist  of  more  than  a  single  individual  sub- 
stance. The  so-called  serum  albumin  may  be  separated  into  at  least 
two  distinct  bodies,  one  capable  of  crystallization,  the  other  an  amor- 
phous body.  The  solution  of  either  of  these  bodies  in  water  gives 
the  ordinary  albumin  reactions.  The  coagulation  temperature  of 
the  serum  albumin  mixture  as  it  occurs  in  serum  or  plasma  varies 
from  700  to  85 °  C.  according  to  the  reaction  of  the  solution  and 
its  content  of  inorganic  material.  Serum  albumin  differs  from  egg 
albumin  in  being  more  lsevorotatory,  in  being  rendered  less  insolu- 
ble by  alcohol,  and  in  the  fact  that  when  precipitated  by  hvdro- 
chloric  acid  it  is  more  easily  soluble  in  an  excess  of  the  reagent. 


184  PHYSIOLOGICAL    CHEMISTRY. 

When  blood  coagulates  and  the  usual  clot  forms,  a  light  yellow 
fluid  exudes.  This  is  blood  serum.  It  differs  from  blood  plasma 
in  containing  a  large  amount  of  fibrin  ferment,  a  body  of  great 
importance  in  the  coagulation  of  the  blood,  and  also  in  possessing 
a  lower  protein  content.  The  protein  material  present  in  plasma 
and  not  found  in  serum  is  the  fibrinogen  which  is  transformed  into 
fibrin  in  the  process  of  coagulation  and  removed.  The  specific 
gravity  of  the  serum  of  human  blood  varies  between  1.026  and 
1.032.  If  blood  be  drawn  into  a  vessel  and  allowed  to  remain  with- 
out stirring  or  agitation  of  any  sort  the  major  portion  of  the  red 
corpuscles  will  sink  away  from  the  upper  surface,  causing  this 
portion  of  the  clot  to  assume  a  lighter  color  due  to  the  predomin- 
ance of  leucocytes.  This  light-colored  portion  of  the  clot  is  called 
the  "buffy  coat." 

Beside  the  protein  constituents  already  mentioned,  other  bodies 
which  are  found  in  both  the  plasma  and  serum  are  the  following: 
Sugar  (dextrose),  fat,  enzymes,  lecithin,  cholesterol  and  its  esters, 
gases,  coloring-matter  (lutein  or  lipochrome)  and  mineral  sub- 
stances. In  addition  to  these  bodies  the  following  substances  have 
been  detected  in  normal  human  blood :  Creatine,  carbamic  acid,  hip- 
puric  acid,  paralactic  acid,  urea  and  uric  acid  {urates).  Some  of 
the  pathological  constituents  of  blood  are  proteoses,  leucine,  tyrosine 
and  other  amino  acids,  biliary  constituents  and  purine  bodies. 

There  has  recently  been  considerable  controversy  regarding  the 
form  of  the  erythrocytes  or  red  blood  corpuscles  of  human  blood. 
It  is  claimed  by  some  investigators  that  the  cells  are  bell-shaped  or 
cup-shaped.  As  the  erythrocytes  occur  normally  in  the  circulation, 
however,  they  are  probably  thin,  non-nucleated,  biconcave  discs. 
When  examined  singly  under  the  microscope,  they  possess  a  pale 
greenish-yellow  color  (see  Plate  IV,  opposite),  whereas  when 
grouped  in  large  masses  a  reddish  tint  is  noted. 

The  blood  of  most  mammals  contains  erythrocytes  similar  in 
form  to  those  of  human  blood.  In  the  blood  of  birds,  fishes,  am- 
phibians and  reptiles  the  erythrocytes  are  ordinarily  more  or  less 
elliptical,  biconvex  and  possess  a  nucleus.  The  erythrocytes  vary 
in  size  with  the  different  animals.  The  average  diameter  of  the 
erythrocytes  of  blood  from  various  species  is  given  in  the  follow- 
ing table  ■} 

1  Wormley's  Micro-Chemistry  of  Poisons,  second  edition,  p.  733. 


PLATE  IV. 


Normal  Erythrocytes  and  Leucocytes. 


BLOOD.  I  <S  5 

Elephant ■,  fa  s  of  an  inch. 

Guinea-pig rfog  of  an  inch. 

Mail niVo  of  an  inch. 

Monkey j  A  z   of  an  inch. 

Dog i  s"sr  of  an  inch. 

Rat •TtsVz  of  an  inch. 

Rabbit $?hs  of  an  inch. 

Mouse sVr.i   of  an  inch. 

Lion fTis  of  an  inch. 

Ox f-Jj.j  of  an  inch. 

Horse T2\7  of  an  inch. 

Pig tzVs   of  an  inch. 

Cat tsts  OI  an  inch. 

Sheep jtjxj  of  an  inch. 

Goat stts  of  an  inch. 

Musk-deer tz^ts  of  an  inch. 

The  erythrocytes  from  whatever  source  obtained,  consist  essen- 
tially of  two  parts,  the  stroma  or  protoplasmic  tissue  and  its  en- 
closed pigment,  hcemoglobin.  For  human  blood  the  number  of 
erythrocytes  present  in  the  fluid  as  obtained  from  well-developed 
males  in  good  physical  condition  is  about  5,500,000  per  cubic  milli- 
meter.1 The  normal  content  of  the  blood  of  adult  females  is  from 
4,000,000  to  4,500,000  per  cubic  millimeter.  The  number  of  ery- 
throcytes varies  greatly  under  different  conditions.  For  instance 
the  number  may  be  increased  after  the  transfusion  of  blood  of  the 
same  species  of  animal ;  by  residing  in  a  high  altitude ;  or  as  a  re- 
sult of  strenuous  physical  exercise  continued  over  a  short  period  of 
time.  An  increase  is  also  noted  in  starvation ;  after  partaking  of 
food ;  after  cold  or  hot  baths ;  after  massage,  as  well  as  after  the 
administration  of  certain  drugs  and  accompanying  certain  diseases 
such  as  cholera,  diarrhoea,  dysentery  and  yellow  atrophy  of  the  liver. 
A  decrease  in  the  number  occurs  in  the  different  forms  of  anaemia. 
The  number  has  been  known  to  increase  to  7,040,000  per  cubic 
millimeter  as  a  result  of  physical  exercise,  while  11,000,000  per 
cubic  millimeter  have  been  noted  in  cases  of  polycythemia  and  in- 
creases nearly  as  great  in  cyanosis.  The  number  has  been  known 
to  decrease  to  500,000  per  cubic  millimeter  or  lower  in  pernicious 
ansemia. 

Oxyhemoglobin,  the  coloring  matter  of  the  blood,  is  a  conju- 
gated protein.  Through  treatment  with  hydrochloric  acid  it  may 
be  split  into  a  protein  body  called  globiii,  and  liccmochroDiogcn,  an 

1  This  statement  is  based  upon  observations  made  upon  the  blood  of  athletes 
in  training.  It  is  generally  stated  in  text-books  that  the  blood  of  males  contains 
about  5,000,000  per  cubic  millimeter. 


i86 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  so. 


1 


OXYHEMOGLOBIN    CRYSTALS    FROM    BLOOD    OF    THE    GUINEA    PlG. 

Reproduced    from    a    micro-photograph    furnished   by    Prof.    E.   T.    Reichert,    of   the 
University   of   Pennsylvania. 


Fig.  51. 


Oxyhemoglobin   Crystals  from  Blood  of  the  Rat. 

Reproduced    from    a    micro-photograph    furnished    by    Prof.    E.    T.    Reichert,    of    the 
University  of   Pennsylvania. 


BLOOD. 


.87 


Oxyhemoglobin  Crystals  from  Blood  of  the  Horse. 

Reproduced    from   a  micro-photograph    furnished   by    Prof.    E.    T.    Reichert,    of    the 
University   of   Pennsylvania. 


Fig.  S3- 


-i 


Oxyhemoglobin  Crystals  from  Blood  of  the  Squirrel. 

Reproduced    from    a    micro-photograph    furnished    by    Prof.    E.    T.    Reichert,    of   the 
University   of   Pennsylvania. 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  54. 


Oxyhemoglobin   Crystals   from   Blood  of  the   Dog. 

Reproduced    from    a   micro-photograph    furnished   by    Prof.    E.    T.    Reichert,    of    the 
University  of  Pennsylvania. 


Fig.  55. 


Oxyhemoglobin   Crystals   from   Blood   of  the   Cat. 

Reproduced    from    a   micro-photograph    furnished   by    Prof.    E.    T.    Reichert,    of   the 
University   of   Pennsylvania. 


BLOOD. 

Fig.  56. 


189 


Oxyhemoglobin  Crystals  from  Blood  of  the  Necturus. 

Reproduced    from    a   micro-photograph    furnished   by    Prof.    E.    T.    Reichert,    of    the 
University   of   Pennsylvania.1 

iron-containing  pigment.  The  latter  body  is  rapidly  transformed 
into  hcematin  in  the  presence  of  oxygen  and  this  in  turn  gives  place 
to  haematin-hydrochloride  or  hcemin  (Figs.  58  and  59,  page  198). 
The  pigment  of  arterial  blood  is  for  the  most  part  loosely  combined 
with  oxygen  and  is  termed  o.ryhaemoglobin,  whereas  the  pigment 
of  venous  blood  is  principally  haemoglobin  (so-called  reduced 
haemoglobin).  Oxyhaemoglobin  is  the  oxygen-carrier  of  the  body 
and  belongs  to  the  class  of  bodies  known  as  respiratory  pigments. 
It  is  held  within  the  stroma  of  the  erythrocyte.  The  reduction  of 
oxyhaemoglobin  to  form  haemoglobin  (so-called  reduced  haemo- 
globin) occurs  in  the  capillaries.  Oxyhaemoglobin  may  be  crys- 
tallized and  a  specific  form  of  crystal  obtained  from  the  blood  of 
each  individual  species  (see  Figs.  50  to  56,  pages  186  to  189).  This 
fact  seems  to  indicate  that  there  are  many  varieties  of  oxyhaemo- 
globin. The  interesting  findings  of  Reichert  and  Brown  are  of 
great  value  in  this  connection.  These  investigators  prepared  oxy- 
haemoglobin crystals  from  over  one  hundred  species  of  animal  and 
subsequently  studied  the  characteristics  of  the  crystals  very  min- 
utely from  the  standpoint  of  crystallography.  Their  findings  may 
prove  of  importance  from  the  standpoint  of  heredity  and  the  origin 
of  species.     They  emphasize  the  following  facts  : 

1  The  micro-photographs  of  oxyhemoglobin  (see  pages  186-189)  and  hasmin 
(see  page  198)  are  reproduced  through  the  courtesy  of  Professors  E.  T. 
Reichert  and  Amos  P.  Brown,  of  the  University  of  Pennsylvania,  who  are 
investigating  the  crystalline  forms  of  biochemic  substances. 


I9O  PHYSIOLOGICAL    CHEMISTRY. 

1.  Crystals  from  all  species  of  a  certain  genus  have  certain 
characteristics  in  general.  Crystals  from  different  genera  however 
exhibit  marked  differences  in  system,  axial  ratios,  etc. 

2.  Crystals  of  different  species  of  a  genus  may  generally  be 
differentiated   by   difference   in    the   angles. 

3.  The  oxyhemoglobin  of  some  species  crystallizes  in  several 
types  of  crystals  in  the  same  preparation.  Generally  the  crystals 
first  formed  belong  to  a  system  of  a  lower  grade  of  symmetry  than 
those  formed  later.  When  such  different  types  of  crystals  occur 
they  may  be  arranged  in  isomorphous  series. 

4.  Certain  definite  angles  recur  in  the  crystals  from  the  blood 
of  various  species  of  animal,  although  the  zoological  connection 
may  be  remote  and  the  crystals  belong  to  different  systems. 

5.  The  constant  recurrence  of  certain  types  of  "twinning" 
in  all  the  crystalline  forms  was  observed. 

6.  Differences  have  been  observed  in  the  crystalline  form  of 
oxyhemoglobin  and  haemoglobin  from  the  blood  of  the  same 
species  in  certain  cases. 

The  following  bodies  may  be  derived  from  haemoglobin,  and  each 
possesses  a  specific  spectrum  which  serves  as  an  aid  in  its  detection 
and  identification :  Oxyhemoglobin,  methaemoglobin,  carbon-mon- 
oxide haemoglobin,  nitric-oxide  haemoglobin,  haemochromogen, 
haematin,  acid-haematin,  alkali-haematin  and  haematoporphyrin  (see 
Absorption  Spectra,  Plates  I  and  II). 

The  white  corpuscles  (or  leucocytes)  of  human  blood  differ  from 
the  red  corpuscles  (or  erythrocytes)  in  many  particulars,  such  as 
being  somewhat  larger  in  size,  in  containing  at  least  a  single  nucleus 
and  in  possessing  amoeboid  movement  (see  Plate  IV,  opposite  page 
184).  They  are  typical  animal  cells  and  therefore  contain  the  fol- 
lowing bodies  which  are  customarily  present  in  such  cells :  Proteins, 
fats,  carbohydrates,  lecithin,  cholesterol,  inorganic  salts  and  water. 
The  normal  number  of  leucocytes  in  human  blood  varies  between 
5,000  and  10,000  per  cubic  millimeter.  The  ratio  between  the  leu- 
cocytes and  erythrocytes  is  about  1 :  350-500.  A  leucocytosis  is 
said  to  exist  when  the  number  of  leucocytes  is  increased  for  any 
reason.  Leucocytoses  may  be  divided  into  two  general  classes,  the 
physiological  and  the  pathological.  Under  the  physiological  form 
would  be  classed  those  leucocytoses  accompanying  pregnancy,  par- 
turition and  digestion,  as  well  as  those  due  to  mechanical  and  ther- 
mal influences.  The  leucocytoses  spoken  of  as  pathological  are  the 
inflammatory,    infectious,    post-haemorrhagic,    toxic    and    experi- 


BLOOD.  191 

mental  forms  as  well  as  the  type  of  leucocytosis  which  accompanies 
malignant  disease. 

The  blood  plates  (platelets  or  plaques)  are  round  or  oval,  color- 
less discs  which  possess  a  diameter  about  one-third  as  great  as  that 
of  the  erythrocytes.  Upon  treatment  with  certain  reagents,  e.  g., 
artificial  gastric  juice,  they  may  be  separated  into  a  homogeneous, 
non-refractive  portion  and  a  granular,  refractive  portion.  The 
blood  plates  are  probably  associated  in  some  way  with  the  coagula- 
tion of  the  blood.  This  relationship  is  not  well  understood  at 
present. 

The  hsemoconein  or  so-called  "  blood  dust  "  is  made  up  of  round 
granules  which  usually  have  a  diameter  somewhat  less  than  one 
micron.  The  serum  of  normal  as  well  as  of  pathological  blood 
contains  these  granules.  They  were  first  described  by  Miiller  to 
whom  they  appeared  as  highly  refractile  granules  possessed  of 
Brownian  movement.  The  "  blood  dust  "  is  apparently  not  con- 
cerned with  the  coagulation  of  the  blood.  The  granules  are  insol- 
uble in  alcohol,  ether  and  acetic  acid  and  are  not  blackened  by  osmic 
acid.  According  to  Miiller  the  granules  making  up  the  so-called 
"  blood  dust  "  constitute  a  new  organized  constituent  of  the  blood, 
whereas  other  investigators  believe  them  to  be  merely  free  granules 
from  certain  of  the  forms  of  leucocytes.  In  common  with  blood 
plates  the  "blood  dust"  possesses  no  clinical  significance. 

The  processes  involved  in  the  coagulation  of  the  blood  are  not 
fully  understood.  Several  theories  have  been  advanced  and  each  has 
its  adherents.  The  theory  which  appears  to  be  fully  as  firmly 
founded  upon  experimental  evidence  as  any  is  the  following :  Blood 
contains  a  zymogen  called  prothrombin  which  combines  with  the 
calcium  salts  present  to  form  an  enzyme  known  as  thrombin  or 
fibrin-ferment.  When  freshly  drawn  blood  comes  in  contact  with 
the  air  the  fibrin-ferment  at  once  acts  upon  the  fibrinogen  present 
and  gives  rise  to  the  formation  of  fibrin.  This  fibrin  forms  in 
shreds  throughout  the  blood  mass  and.  holding  the  form  elements 
of  the  blood  within  its  meshes,  serves  to  produce  the  typical  blood 
clot.  The  fibrin  shreds  gradually  contract,  the  whole  clot  assumes 
a  jelly-like  appearance  and  the  yellowish  serum  exudes.  If,  im- 
mediately upon  the  withdrawal  of  blood  from  the  body,  the  fluid 
be  rapidly  stirred  or  thoroughly  "  whipped  "  with  a  bundle  of 
coarse  strings,  twigs  or  a  specially  constructed  beater,  the  fibrin 
shreds  will  not  form  in  a  network  throughout  the  blood  mass  but 
instead  will  cling  to  the  device  used  in  beating.     In  this  way  the 


I92  PHYSIOLOGICAL    CHEMISTRY. 

fibrin  may  be  removed  and  the  remaining  fluid  is  termed  defibrin- 
ated  blood.  The  above  theory  of  the  coagulation  of  the  blood  may 
be  stated  briefly  as  follows : 

I.  Prothrombin  -f-  Calcium  Salts  =  Thrombin  (or  Fibrin- 
ferment). 

II.  Thrombin  (or  Fibrin-ferment)   +  Fibrinogen  =  Fibrin. 
Among   the   medico-legal   tests    for   blood   are   the    following: 

(1)  Microscopical  identification  of  the  erythrocytes,  (2)  spectro- 
scopic identification  of  blood  solutions,  (3)  the  guaiac  test,  (4) 
the  benzidine  reaction,  (5)  preparation  of  hsemin  crystals.  Of 
these  five  tests  the  two  last  named  are  generally  considered  to  be  the 
most  satisfactory.  They  give  equally  reliable  results  with  fresh 
blood  and  with  blood  from  clots  or  stains  of  long  standing,  pro- 
vided the  latter  have  not  been  exposed  to  a  high  temperature, 
or  to  the  rays  of  the  sun  for  a  long  period.  The  technique  of  the 
tests  is  simple  and  the  formation  of  the  dark  brown  or  chocolate 
colored  crystals  of  haemin  or  the  production  of  the  green  or  blue 
color  with  benzidine  is  indisputable  proof  of  the  presence  of  blood  in 
the  fluid,  clot  or  stain  examined.  The  weak  point  of  the  tests, 
medico-legally,  lies  in  the  fact  that  they  do  not  differentiate  between 
human  blood  and  that  of  certain  other  species  of  animal. 

The  guaiac  test  (see  page  196),  although  generally  considered 
less  accurate  than  the  haemin  test,  is  really  a  more  delicate  test 
than  the  hsemin  test  if  properly  performed.  One  of  the  most 
common  mistakes  in  the  manipulation  of  this  test  is  the  use  of  a 
guaiac  solution  which  is  too  concentrated  and  which,  when  brought 
into  contact  with  the  aqueous  blood  solution,  causes  the  separation 
of  a  voluminous  precipitate  of  a  resinous  material  which  may  ob- 
scure the  blue  coloration :  this  is  particularly  true  of  the  test  when 
used  for  the  examination  of  blood  stains.  A  solution  of  guaiac 
made  by  dissolving  1  gram  of  the  resin  in  60  c.c.  of  95  per  cent 
alcohol  is  very  satisfactory  for  general  use.  The  test  is  frequently 
objected  to  upon  the  ground  that  various  other  substances,  e.  g., 
milk,  pus,  saliva,  etc.,  respond  to  the  test  and  that  it  cannot  there- 
fore be  considered  a  specific  test  for  blood  and  is  of  value  only  in 
a  negative  sense.  We  have  demonstrated  to  our  own  satisfaction, 
however,  that  milk  many  times  gives  the  blue  color  upon  the  addi- 
tion of  an  alcoholic  solution  of  guaiac  resin  without  the  addition  of 
hydrogen  peroxide  or  old  turpentine.  Buckmaster  has  very  recently 
advocated  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead 
of  an  alcoholic  solution  of  guaiac  resin.     He  claims  that  he  was 


BLOOD.  193 

able  to  produce  the  blue  color  upon  the  addition  of  the  guaiaconic 
acid  to  milk  only  when  the  sample  of  milk  tested  was  brought  from 
the  country  in  sterile  hollies,  and  further,  that  no  sample  of  London 
milk  which  he  examined  responded  to  the  test.  In  the  application 
of  the  guaiac  test  to  the  detection  of  blood,  he  states  that  he  was 
able  to  detect  laked  blood  when  present  in  the  ratio  1 :  5,000,000 
and  unlaked  blood  when  present  in  the  ratio  1  :  1,000,000.  This 
author  considers  the  guaiac  test  to  be  far  more  trustworthy  than 
is  generally  believed. 

Up  to  within  very  recent  times  it  has  been  impossible  to  make  an 
absolute  differentiation  of  human  blood.  Recently,  however,  the 
so-called  "biological"  blood  test  has  made  such  a  differentiation 
possible.  This  test,  known  as  the  Bordet  reaction,  is  founded  upon 
the  fact  that  the  blood  serum  of  an  animal  into  which  has  been 
injected  the  blood  of  another  animal  of  different  species  develops 
the  property  of  agglutinating  and  dissolving  erythrocytes  similar 
to  those  injected,  but  exerts  this  influence  upon  the  blood  from  no 
other  species.  The  antiserum  used  in  this  test  is  prepared  by  inject- 
ing rabbits  with  5-10  c.c.  of  human  defibrinated  blood,  at  intervals 
of  about  four  days  until  a  total  of  between  50  and  80  c.c.  has  been 
injected.  After  a  lapse  of  one  or  two  weeks  the  animal  is  bled, 
the  serum  collected,  placed  in  sterile  tubes  and  preserved  for  use 
as  needed.  In  examining  any  specific  solution  for  human  blood 
it  is  simply  necessary  to  combine  the  antiserum  and  the  solution 
under  examination  in  the  proportion  of  1  :  100  and  place  the  mix- 
ture at  370  C.  If  human  blood  is  present  in  the  solution  a  turbidity 
will  be  noted  and  this  will  change  within  three  hours  to  a  dis- 
tinctly flocculent  precipitate.  This  antiserum  will  react  thus  with 
no  other  known  substance. 

Experiments  on  Blood. 

I.  Defibrinated  Ox-blood. 

i.  Reaction. — Moisten  red  and  blue  litmus  papers  with  10  per 
cent  sodium  chloride  solution  and  test  the  reaction  of  the  defibri- 
nated blood. 

2.  Microscopical  Examination. — Examine  a  drop  of  defibri- 
nated blood  under  the  microscope.  Compare  the  objects  you  ob- 
serve with  Plate  IV,  opposite  page  184.  Repeat  the  test  with  a 
drop  of  your  own  blood. 

3.  Specific  Gravity. — Determine  the  specific  gravity  of  defibri- 
nated blood  by  means  of  an  ordinary  specific  gravity  spindle.     Com- 

M 


194  PHYSIOLOGICAL    CHEMISTRY. 

pare  this  result  with  the  specific  gravity  as  determined  by  Hammer- 
schlag's  method  in  the  next  experiment. 

4.  Specific  Gravity  by  Hammerschlag's  Method. — Fill  an 
ordinary  urinometer  cylinder  about  one-half  full  of  a  mixture  of 
chloroform  and  benzene,  having  a  specific  gravity  of  approximately 
1.050.  Into  this  mixture  allow  a  drop  of  the  blood  under  exami- 
nation to  fall  from  a  pipette  or  directly  from  the  finger  in  case 
fresh  blood  is  being  examined.  Care  must  be  taken  not  to  use 
too  large  a  drop  of  blood  and  to  keep  the  drop  from  coming  in 
contact  with  the  walls  of  the  cylinder.  If  the  blood  drop  sinks  to 
the  bottom  of  the  vessel,  thus  showing  it  to  be  of  higher  specific 
gravity  than  the  surrounding  fluid,  add  chloroform  until  the  blood 
drop  remains  suspended  in  the  mixture.  Stir  carefully  with  a  glass 
rod  after  adding  the  chloroform.  If  the  blood  drop  rises  to  the 
surface  upon  being  introduced  into  the  mixture,  thus  showing  it  to 
be  of  lower  specific  gravity  than  the  surrounding  fluid,  add  benzene 
until  the  blood  drop  remains  suspended  in  the  mixture.  Stir  with 
a  glass  rod  after  the  benzene  is  added.  After  the  blood  drop  has 
been  brought  to  a  suspended  position  in  the  mixture  by  means  of 
one  or  more  additions  of  chloroform  and  benzene  this  final  mixture 
should  be  filtered  through  muslin  and  its  specific  gravity  accurately 
determined.  What  is  the  specific  gravity  of  the  blood  under  ex- 
amination? 

5.  Tests  for  Various  Constituents. — Place  10  c.c.  of  defibri- 
nated  blood  in  an  evaporating'  dish,  dilute  with  100  c.c.  of  water 
and  heat  to  boiling.  Is  there  any  coagulation,  and  if  so  what 
bodies  form  the  coagulum?  At  the  boiling-point  acidulate  slightly 
with  dilute  acetic  acid.  Filter.  The  filtrate  should  be  clear  and 
the  coagulum  dark  brown.  Reserve  this  coagulum.  What  body 
gives  the  coagulum  this  color?  Evaporate  the  filtrate  to  about 
25  c.c,  filtering  off  any  precipitate  which  may  form  in  the  process.' 
Make  the  following  tests  upon  the  filtrate : 

(a)  Fehling's  Test. — Test  for  sugar  according  to  directions 
given  on  page  27. 

(b)  Chlorides. — To  a  small  amount  of  the  filtrate  in  a  test-tube 
add  a  few  drops  of  nitric  acid  and  a  little  argentic  nitrate.  In  the 
presence  of  chloride,  a  white  precipitate  of  argentic  chloride  will 
form. 

(c)  Phosphates. — Test  for  phosphates  by  nitric  acid  and  molyb- 
dic  solution  according  to  directions  given  on  page  57. 

(d)  Proteose  and  Peptone. — Test  a  small  amount  of  the  solu- 


BLOOD.  195 

tion  for  proteose  and  peptone  by  saturating"  with  ammonium  sul- 
phate according"  to  directions  given  on  page  114. 

(c)  Crystallization  of  Sodium  Chloride. — Place  the  remainder 
of  the  filtrate  in  a  watch  glass  and  evaporate  it  on  a  water-bath. 
Examine  the  crystals  under  the  microscope  and  compare  them  with 
those  in  Fig.  60,  page  200. 

6.  Test  for  Iron. — Incinerate  a  small  portion  of  the  coagulum 
from  the  last  experiment  (5)  in  a  porcelain  crucible.  Cool,  dis- 
solve the  residue  in  dilute  hydrochloric  acid  and  test  for  iron  by 
potassium  ferrocyanide  or  ammonium  thiocyanate.  Which  of 
the  constituents  of  the  blood  contains  the  iron? 

7.  Laky  Blood.  —  Note  the  opacity  of  ordinary  defibrinated 
blood.  Place  a  few  cubic  centimeters  of  this  blood  in  a  test-tube 
and  add  water,  a  little  at  a  time,  until  the  blood  is  rendered  trans- 
parent. It  is  now  laky  blood.  How  does  the  water  act  in  causing 
this  transparency?  Examine  a  drop  of  laky  blood  under  the  micro- 
scope. How  does  its  microscopical  appearance  differ  from  that  of 
unaltered  blood?  What  other  agents  may  be  used  to  render  blood 
laky? 

Fig.   57. 


Effect  of  Water  on  Erythrocytes. 

8.  Osmotic  Pressure. — Place  a  few  cubic  centimeters  of  blood 
in  each  of  three  test-tubes.  Lake  the  blood  in  the  first  tube  accord- 
ing to  directions  given  in  the  last  experiment  (/)  :  add  an  equal 
volume  of  isotonic  (0.9  per  cent)  sodium  chloride  to  the  blood  in 
the  second  tube,  and  an  equal  volume  of  10  per  cent  sodium  chloride 
to  the  blood  in  the  third  tube.  Mix  thoroughly  by  shaking  and 
after  a  few  moments  examine  a  drop  from  each  of  the  three  tubes 


I96  PHYSIOLOGICAL    CHEMISTRY. 

under  the  microscope  (see  Figs.  57  and  115,  pages  195  and  359). 
What  do  you  find  and  what  is  your  explanation  from  the  stand- 
point of  osmotic  pressure? 

9.  Agglutination.— To  about  5  c.c.  of  a  dilute  sodium  chloride 
solution  of  ricin1  in  a  test-tube  add  about  one-half  cubic  centimeter 
of  defibrinated  blood  and  shake  the  mixture  thoroughly.  Allow  the 
tube  to  stand  about  15  minutes  and  examine  a  drop  of  the  contents 
under  the  microscope.  Note  the  "  clumping  "  or  "  agglutination  " 
of  the  erythrocytes,  and  contrast  this  phenomena  with  the  appear- 
ance of  normal  blood  as  just  examined  in  experiment  8. 

10.  Diffusion  of  Haemoglobin.  — Prepare  some  laky  blood,  thus 
liberating  the  haemoglobin  from  the  erythrocytes.  Test  the  dif- 
fusion of  the  haemoglobin  by  preparing  a  dialyzer  like  one  of  the 
models  shown  in  Fig.  1,  page  25.  How  does  haemoglobin  differ 
from  other  well-known  crystallizable  bodies? 

11.  Guaiac  Test. — To  5  c.c.  of  water  in  a  test-tube  add  two 
drops  of  blood.  By  means  of  a  pipette  drop  an  alcoholic  solution 
of  guaiac  (strength  about  1  :6o)2  into  the  resulting  mixture  until 
a  turbidity  is  observed  and  add  old  turpentine  or  hydrogen  perox- 
ide, drop  by  drop,  until  a  blue  color  is  obtained.  Do  any  other  sub- 
stances respond  in  a  similar  manner  to  this  test?  Is  a  positive 
guaiac  test  a  sure  indication  of  the  presence  of  blood? 

12.  Schumm's  Modification  of  the  Guaiac  Test.— To  about 
5  c.c.  of  the  solution  under  examination3  in  a  test-tube  add  about 
ten  drops  of  freshly  prepared  alcoholic  solution  of  guaiac.  Agitate 
the  tube  gently,  add  about  20  drops  of  old  turpentine,  subject  the 
tube  to  a  thorough  shaking  and  permit  it  to  stand  for  about  2-3 
minutes.  A  blue  color  indicates  the  presence  of  blood  in  the  solu- 
tion under  examination.  In  case  there  is  insufficient  blood  to  yield 
a  blue  color  under  these  conditions,  a  few  c.c.  of  alcohol  should  be 
added  and  the  tube  gently  shaken,  whereupon  a  blue  coloration 
will  appear  in  the  upper  alcohol-turpentine  layer. 

A  control  test  should  always  be  made,  using  water  in  place  of  the 
solution  under  examination.  In  the  detection  of  very  minute  traces 
of  blood  only  3-5  drops  of  the  guaiac  solution  should  be  employed. 

13.  Adler's  Benzidine  Reaction. — This  is  one  of  the  most  deli- 
cate of  the  reactions  for  the  detection  of  blood.     Different  benzi- 

1  A  protein  constituent  of  the  castor  bean. 

2  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead 
of  an  alcoholic  solution  of  guaiac  resin. 

s  Alkaline  solutions  should  be  made  slightly  acid  with  acetic  acid,  as  the  blue 
end-reaction  is  very  sensitive  to  alkali. 


BLOOD.  197 

dine  preparations  vary  greatly  in  their  sensitiveness,  however. 
Inasmuch  as  benzidine  solutions  change  readily  upon  contact  with 
light  it  is  essential  that  they  be  kept  in  a  dark  place.  The  test  is 
performed  as  follows :  To  a  saturated  solution  of  benzidine  in 
alcohol  or  glacial  acetic  acid  add  an  equal  volume  of  3  per  cent 
hydrogen  peroxide  and  one  c.c.  of  the  solution  under  examination. 
If  the  mixture  is  not  already  acid  render  it  so  with  acetic  acid, 
and  note  the  appearance  of  a  green  or  blue  color.  A  control  test 
should  be  made  substituting  water  for  the  solution  under  examina- 
tion. The  sensitiveness  of  the  benzidine  reaction  is  greater  when 
applied  to  aqueous  solutions  than  when  applied  to  the  urine. 

14.  Haemin  Test.— (a)  Teichmann's  Method.  —  Place  a  very 
small  drop  of  blood  on  a  microscopic  slide,  add  a  minute  grain 
of  sodium  chloride1  and  carefully  evaporate  to  dryness  over  a  low 
flame.  Put  a  cover  glass  in  place,  run  underneath  it  a  drop  of 
glacial  acetic  acid  and  warm  gently  until  the  formation  of  gas 
bubbles  is  noted.  Add  another  drop  of  glacial  acetic  acid,  cool 
the  preparation,  examine  under  the  microscope  and  compare  the 
crystals  with  those  shown  in  Figs.  58  and  59,  page  198.  The  haemin 
crystals  result  from  the  decomposition  of  the  haemoglobin  of  the 
blood.  What  are  the  steps  involved  in  this  process  ?  The  haemin 
crystals  are  also  called  Teichmann's  crystals.  Is  this  an  absolute 
test  for  blood  ?  Is  it  possible  to  differentiate  between  human  blood 
and  the  blood  of  other  species  by  means  of  the  haemin  test? 

(b)  Atkinson  and  Kendall's  Method. — Introduce  a  small  amount 
of  the  solution  under  examination  into  a  tube  closed  at  one  end, 
add  sodium  chloride  and  glacial  acetic  acid  as  in  Teichmann's 
method,2  fuse  or  tightly  plug  the  open  end  of  the  tube  and  heat 
for  fifteen  minutes  in  a  boiling  water-bath.3  Remove  the  tube  and! 
permit  it  to  cool  to  room  temperature  spontaneously.  When  the 
tube  has  cooled,  break  it  open,  transfer  the  contents  to  a  watch 
glass  or  small  evaporating  dish  and  concentrate  on  a  water-bath 
until  the  volume  of  the  fluid  in  the  watch  glass  or  dish  has  been 
reduced  to  a  few  drops.  Transfer  a  drop  of  this  fluid  to  a  slide., 
cover  with  a  cover  slip,  allow  the  slide  to  stand  for  a  few  minutes-, 
and  examine  it  under  a  microscope.  Compare  the  crvstals  with 
those  shown  in  Figs.  58  and  59.  page  198.  In  case  crystals  of 
sodium  chloride  (see  Fig.  60,  page  200)   obstruct  the  view  of  the 

1  Buckmaster  considers  the  use  of  potassium  chloride  preferable. 

2  Care  should  be  taken  not  to  add  too  great  an  excess  of  these  reagents. 

3  This  process  insures  constancy  of  temperature  and  strength  of  reagents. 


I98  PHYSIOLOGICAL    CHEMISTRY. 

Fig.  58. 

Hemin  Crystals  from  Human  Blood. 

Reproduced    from    a   micro-photograph    furnished   by    Prof.    E.    T.    Reichert,    of    the 
University  of  Pennsylvania. 


Fig.  59. 


dk  \ 


) ;   ~ux  *.% 


\ 


ELemin  Crystals  from  Sheep  Blood. 


Reproduced    from    a   micro-photograph    furnished   by    Prof.    E.    T.    Reichert,    of    the 
University  of  Pennsylvania. 


BLOOD.  1 99 

hsemin  crystals  dissolve  the   sodium  chloride  crystals  by  running 
a  drop  of  water  under  the  cover  slip. 

(c)  v.  Zeynek  and  Nencki's  Method. — To  10  c.c.  of  defibrinated 
blood  add  acetone  until  no  more  precipitate  forms.  Filter  off  the 
precipitated  protein  and  extract  it  with  10  c.c.  of  acetone  made 
acid  with  2-3  drops  of  hydrochloric  acid.  Place  a  drop  of  the 
resulting  colored  extract  on  a  slide,  immediately  place  a  cover  glass 
in  position  and  examine  under  the  microscope.  Upon  the  evapora- 
tion of  the  acetone,  crystals  of  haemin  will  form.  Larger  crystals 
may  be  obtained  by  evaporating  the  acetone  extract  about  one- 
half,  transferring  it  to  a  stoppered  vessel  and  allowing  it  to  remain 
over  night. 

(d)  Schalfi Jew's  Method. — Place  20  c.c.  of  glacial  acetic  acid 
in  a  small  beaker  and  heat  to  8o°  C.  Add  5  c.c.  of  strained  defibri- 
nated blood,  again  bring  the  temperature  to  8o°  C,  remove  the 
flame  and  allow  the  mixture  to  cool.  Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  reproduced  in  Figs. 
58  and  59,  page  198. 

15.  Catalytic  Action.  — To  about  10  drops  of  blood  in  a  test- 
tube  add  twice  the  volume  of  hydrogen  peroxide,  without  shaking. 
The  mixture  foams.     What  is  the  cause  of  this  phenomenon? 

16.  Preparation  of  Haematin. — Place  100  c.c.  of  laked  blood 
in  a  beaker  and  add  95  per  cent  alcohol  until  precipitation  ceases. 
What  bodies  are  precipitated?  Transfer  the  precipitate  to  a  flask 
and  boil  with  95  per  cent  alcohol  previously  acidulated  with  sul- 
phuric acid.  Through  the  action  of  the  acid  the  haemoglobin  is 
split  into  haematin  and  a  protein  body  called  globin.  Later  the 
"sulphuric  acid  ester  of  haematin"  is  formed,  which  is  soluble  in 
the  alcohol.  Continue  heating  until  the  precipitate  is  no  longer 
colored,  then  filter.  Partly  saturate  the  filtrate  with  sodium  chlo- 
ride and  warm.  Iri  this  process  the  "  hydrochloric  acid  ester  of 
haematin  "  is  formed.  Filter  and  dissolve  on  the  filter  paper  by 
sodium  carbonate.  Save  this  alkaline  solution  of  haematin  and 
make  a  spectroscopic  examination  later  after  becoming  familiar 
with  the  use  of  the  spectroscope.  How  does  the  spectrum  of  oxy- 
hemoglobin differ  from  that  of  the  derived  alkali  hcematin? 

17.  Variation  in  Size  of  Erythrocytes.  — Prepare  two  small 
funnels  with  filter  papers  such  as  are  used  in  quantitative  analysis. 
Moisten  each  paper  with  normal  (isotonic)  salt  solution.  Into  one 
funnel  introduce  a  small  amount  of  defibrinated  ox  blood  and  into 
the  other  funnel  allow  blood  to  drop  directly  from  a  decapitated 


200 


PHYSIOLOGICAL    CHEMISTRY. 


frog.  Note  that  the  filtrate  from  the  ox  blood  is  colored  whereas 
that  from  the  frog  blood  is  colorless.  What  deduction  do  you 
make  regarding  the  relative  size  of  the  erythrocytes  in  ox  and 
frog  blood?     Does  either  filtrate  clot?     Why? 

II.  Blood  Serum. 

1.  Coagulation  Temperature. — Place  5  c.c.  of  undiluted  serum 
in  a  test-tube  and  determine  its  temperature  of  coagulation  accord- 
ing to  the  method  described  on  page  100.  Note  the  temperature  at 
which  a  cloudiness  occurs  as  well  as  the  temperature  at  which  coag- 
ulation is  complete. 

2.  Precipitation  by  Alcohol. — To  5  c.c.  of  serum  in  a  test-tube 
add  twice  the  amount  of  95  per  cent  alcohol  and  thoroughly  mix 
by  shaking.  What  is  this  precipitate?  Make  a  confirmatory  test. 
Test  the  alcoholic  filtrate  for  protein.    Explain  the  result. 

3.  Proteins  of  Blood  Serum. — Place  about  20  c.c.  of  undiluted 
serum  in  a  small  evaporating  dish,  heat  to  boiling  and  at  the  boiling- 


Fig.  60. 


Sodium   Chloride. 


point  acidify  slightly  with  dilute  acetic  acid.  Of  what  does  this 
coagulum  consist?  Filter  off  the  coagulum  (reserve  the  filtrate) 
and  test  it  as  follows: 

(a)  Millon's  Reaction. — Make  the  test  according  to  directions 
given  on  page  90. 

(b)  Hopkins-Cole  Reaction. — Make  the  test  according  to  direc- 
tions given  on  page  10 1. 


BLOOD.  20 1 

4.  Sugar  in  Serum. — Test  a  little  of  the  filtrate  from  Experi- 
ment 3  by  Fehling's  test.     What  do  you  conclude? 

5.  Detection  of  Sodium  Chloride. —  (a)   Test   a  little  of   the 

filtrate  from  Experiment  3  for  chlorides,  by  the  use  of  nitric  acid 
and  argentic  nitrate,  (b)  Evaporate  5  c.c.  of  the  filtrate  from 
Experiment  3  in  a  watch  glass  on  a  water-bath.  Examine  the 
crystals  and  compare  them  with  those  reproduced  in  Fig.  60, 
page  200. 

6.  Separation  of  Serum  Globulin  and  Serum  Albumin. — Place 
10  c.c.  of  blood  serum  in  a  small  beaker  and  saturate  with  magne- 
sium sulphate.  What  is  this  precipitate?  Filter  it  off  and  acidify 
the  filtrate  slightly  with  acetic  acid.  What  is  this  second  precipi- 
tate? Filter  this  precipitate  off  and  test  the  filtrate  by  the  biuret 
test.     What  do  you  conclude? 

III.  Blood  Plasma. 

1.  Preparation  of  Oxalated  Plasma. — Allow  arterial  blood  to 
run  into  an  equal  volume  of  0.2  per  cent  ammonium  oxalate 
solution. 

2.  Preparation  of  Fibrinogen. — To  2j  c.c.  of  oxalated  plasma 
add  an  equal  volume  of  saturated  sodium  chloride  solution.  Xote 
the  precipitation  of  fibrinogen.  Filter  off  the  precipitate  (reserve 
the  filtrate)  and  test  it  by  a  protein  color  test  (see  page  90). 

3.  Effect  of  Calcium  Salts. — Place  a  small  amount  of  oxalated 
plasma  in  a  test-tube  and  add  a  few  drops  of  a  2  per  cent  calcium 
chloride  solution.     What  occurs?     Explain  it. 

4.  Preparation  of  Salted  Plasma. — Allow  arterial  blood  to  run 
into  an  equal  volume  of  a  saturated  solution  of  sodium  sulphate 
or  a  10  per  cent  solution  of  sodium  chloride.  Keep  the  mixture 
in  a  cold  place  for  about  twenty-four  hours. 

5.  Effect  of  Dilution. — Place  a  few  drops  of  salted  plasma  in  a 
test-tube  and  dilute  it  with  10-15  volumes  of  water.  What  do  you 
observe?     Explain  it. 

6.  Crystallization  of  Oxyhemoglobin. — Reicherfs  Method. — 
Add  to  5  c.c.  of  the  blood  of  the  dog,  horse,  guinea-pig,  or  rat, 
before  or  after  laking,  or  defibrinating,  from  1  to  5  per  cent  of 
ammonium  oxalate  in  substance.  Place  a  drop  of  this  oxalated 
blood  on  a  slide  and  examine  under  the  microscope.  The  crystals 
of  oxyhemoglobin  will  be  seen  to  form  at  once  near  the  margin 
of  the  drop,  and  in  a  few  minutes  the  entire  drop  may  be  a  solid 
mass  of  crystals.  Compare  the  crystals  with  those  shown  in  Figs. 
50  to  56,  pages  186  to  189. 


202  PHYSIOLOGICAL    CHEMISTRY. 

IV.  Fibrin. 

1.  Preparation  of  Fibrin. — Allow  blood  to  flow  directly  from 
the  animal  into  a  vessel  and  rapidly  whip  it  by  means  of  a  bundle  of 
twigs,  a  mass  of  strong-  cords  or  a  specially  constructed  beater. 
If  a  pure  fibrin  is  desired  it  is  not  best  to  attempt  to  manipulate 
a  large  volume  of  blood  at  one  time.  After  the  fibrin  has  been 
collected  it  should  be  freed  from  any  adhering  blood  clots  and 
washed  in  water  to  remove  further  traces  of  blood.  The  pure 
product  should  be  very  light  in  color.  It  may  be  preserved  under 
glycerol,   dilute   alcohol   or   chloroform  water. 

2.  Solubility. — Try  the  solubility  of  small  shreds  of  freshly 
prepared  fibrin  in  the  usual  solvents. 

3.  Millon's  Reaction. — Make  the  test  according  to  directions 
given  on  page  90. 

4.  Hopkins-Cole  Reaction. — Make  the  test  according  to  direc- 
tions given  on  page  10 1. 

5.  Biuret  Test. — Make  the  test  according  to  directions  given 

on  page  92. 

V.  Detection  of  Blood  in  Stains  on  Cloth,  etc. 

i.  Identification  of  Corpuscles. — If  the  stain  under  examina- 
tion is  on  cloth  a  portion  should  be  extracted  with  a  few  drops 
of  glycerol  or  normal  (0.9  per  cent)  sodium  chloride  solution.  A 
drop  of  this  solution  should  then  be  examined  under  the  micro- 
scope to  determine  if  corpuscles  are  present. 

2.  Tests  on  Aqueous  Extract. — A  second  portion  of  the  stain 
should  be  extracted  with  a  small  amount  of  water  and  the  follow- 
ing tests  made  upon  the  aqueous  extract : 

(a)  Hcumochromogen. — Make  a  small  amount  of  the  extract  al- 
kaline by  potassium  hydroxide  or  sodium  hydroxide,  and  heat 
until  a  brownish-green  color  results.  Cool  and  add  a  few  drops  of 
ammonium  sulphide  or  Stokes'  reagent  (see  page  203)  and  make  a 
spectroscopic  examination.  Compare  the  spectrum  with  that  of 
hsemochromogen    (see  Absorption  Spectra,   Plate  II). 

(b)  Hcemin  Test. — Make  this  test  upon  a  small  drop  of  the  aque- 
ous extract  according  to  the  directions  given  on  page  197. 

(c)  Guaiac  Test. — Make  this  test  on  the  aqueous  extract  accord- 
ing to  the  directions  given  on  page  196.  The  guaiac  solution  may 
also  be  applied  directly  to  the  stain  without  previous  extraction  in 
the  following  manner :  Moisten  the  stain  with  water,  and  after 
allowing  it  to  stand  several  minutes,  add  an  alcoholic  solution  of 
guaiac   (strength  about  1  :  60)   and  a  little  hydrogen  peroxide  or 


BLOOD.  203 

old  turpentine.     The  customary  blue  color  will  be  observed  in  the 
presence  of  blood. 

(d )  Benzidine  Reaction.  —  Make  this  test  according  to  directions 
given  on  p.  196. 

(e)  Acid  Hccmulin.—  M  the  stain  fails  to  dissolve  in  water  ex- 
tract with  acid  alcohol  and  examine  the  spectrum  for  absorption 
bands  of  acid  haematin  (see  Absorption  Spectra.  Plate  II). 

VI.  Spectroscopic  Examination   of  Blood. 
(For  Absorption  Spectra  see  Plates  I.  and  II.) 

Either  the  angular-vision  spectroscope  (Figs.  62  and  63,  page 
204)  or  the  direct- vision  spectroscope  (Fig.  61,  below)  may  be 
used  in  making  the  spectroscopic  examination  of  the  blood.  For  a 
complete  description  of  these  instruments  the  student  is  referred 
to  any  standard  text-book  of  physics. 

1.  Oxyhaemoglobin. — Examine  dilute  (1  150)  defibrinated  blood 
spectroscopically.  Note  the  broad  absorption-band  between  D  and 
E.  Continue  the  dilution  until  this  single  broad  band  gives  place 
to  two  narrow  bands,  the  one  nearer  the  D  line  being  the  narrower. 
These  are  the  typical  absorption-bands  of  oxyhaemoglobin  obtained 
from  dilute  solutions  of  blood.  Now  dilute  the  blood  very  freely 
and  note  that  the  bands  gradually  become  more  narrow  and,  if  the 
dilution  is  sufficiently  great,  they  finally  entirely  disappear. 

Fig.  61. 


Direct-visiox  Spectroscope. 

2.  Haemoglobin  (so-called  'Reduced  Haemoglobin). — To  blood 
which  has  been  diluted  sufficiently  to  show  well  defined  oxyhaemo- 
globin absorption-bands  add  a  small  amount  of  Stokes'  reagent.1 
The  blood  immediately  changes  in  color  from  a  bright  red  to  violet- 
red.  The  oxyhaemoglobin  has  been  reduced  through  the  action  of 
Stokes'  reagent  and  haemoglobin  (so-called  reduced  haemoglobin) 
has  been  formed.     This  has  been  brought  about  by  the  removal 

1  Stokes'  reagent  is  a  solution  containing  2  per  cent  ferrous  sulphate  and 
3  per  cent  tartaric  acid.  When  needed  for  use  a  small  amount  should  be 
placed  in  a  test-tube  and  ammonium  hydroxide  added  until  the  precipitate 
which  forms  on  the  first  addition  of  the  hydroxide  has  entirely  dissolved.  This 
produces  ammonium  ferrotartrate  which  is  a  reducing  agent. 


204 


PHYSIOLOGICAL    CHEMISTRY. 


of  some  of  the  loosely  combined  oxygen  from  the  oxyhemoglobin. 
Examine  this  haemoglobin  spectroscopically.  Note  that  in  place  of 
the  two  absorption  bands  of  oxyhaemoglobin  we  now  have  a  single 


Fig.  6: 


Angular-vision  Spectroscope  Arranged  for  Absorption  Analysis. 

broad  band  lying  almost  entirely  between  D  and  E.  This  is  the 
typical  spectrum  of  haemoglobin.  If  the  solution  showing  this 
spectrum  be  shaken  in  the  air  for  a  few  moments  it  will  again  as- 
sume the  bright  red  color  of  oxyhemoglobin  and  show  the  char- 
acteristic spectrum  of  that  pigment. 

Fig.  63. 


Diagram  of  Angular-vision   Spectroscope.     (Long.) 

The  white  light  F  enters  the  collimator  tube  through  a  narrow  slit  and  passes  to 
the  prism  P,  which  has  the  power  of  refracting  and  dispersing  the  light.  The  rays 
then  pass  to  the  double  convex  lens  of  the  ocular  tube  and  are  deflected  to  the  eye- 
piece E.  The  dotted  lines  show  the  magnified  virtual  image  which  is  formed.  The 
third  tube  contains  a  scale  whose  image  is  reflected  into  the  ocular  and  shown  with 
the  spectrum.  Between  the  light  F  and  the  collimator  slit  is  placed  a  cell  to  hold 
the  solution  undergoing  examination. 


BLOOD.  205 

3.  Carbon  Monoxide  Haemoglobin. — The  preparation  of  this 

pigment  may  be  easily  accomplished  by  passing  ordinary  illumi- 
nating gas1  through  defibrinated  ox-blood.  Blood  thus  treated 
assumes  a  brighter  tint  (carmine)  than  that  imparted  by  oxy- 
hemoglobin. In  very  dilute  solution  oxyhemoglobin  appears  yel- 
lowish-red whereas  carbon  monoxide  haemoglobin  under  the  same 
conditions  appears  bluish-red.  Examine  the  carbon  monoxide 
hemoglobin  solution  spectroscopically.  Observe  that  the  spectrum 
of  this  body  resembles  the  spectrum  of  oxyhemoglobin  in  showing 
two  absorption-bands  between  D  and  E.  The  bands  of  carbon  mon- 
oxide hemoglobin,  however,  are  somewhat  nearer  the  violet  end  of 
the  spectrum.  Add  some  Stokes'  reagent  to  the  solution  and  again 
examine  spectroscopically.  Note  that  the  position  and  intensity  of 
the  absorption  bands  remain  unaltered. 

The  following  is  a  delicate  chemical  test  for  the  detection  of 
carbon  monoxide  hemoglobin  : 

Tan  11  in  Test.  —  Divide  the  blood  to  be  tested  into  two  portions 
and  dilute  each  with  four  volumes  of  distilled  water.  Place  the 
diluted  blood  mixtures  in  two  small  flasks  or  large  test-tubes  and 
add  20  drops  of  a  10  per  cent  solution  of  potassium  ferricyanide.2 
Allow  both  solutions  to  stand  for  a  few  minutes,  then  stopper  the 
vessels  and  shake  one  vigorously  for  10-15  minutes,  occasionally  re- 
moving the  stopper  to  permit  air  to  enter  the  vessel.3  Add  5-10 
drops  of  ammonium  sulphide  (yellow)  and  10  c.c.  of  a  10  per 
cent  solution  of  tannin  to  each  flask.  The  contents  of  the  shaken 
flask  will  soon  exhibit  the  formation  of  a  dirty  olive-green  precipi- 
tate, whereas  the  flask  which  was  not  shaken  and  which,  therefore, 
still  contains  carbon  monoxide  hemoglobin,  will  exhibit  a  bright 
red  precipitate,  characteristic  of  carbon  monoxide  hemoglobin. 
This  test  is  more  delicate  than  the  spectroscopic  test  and  serves  to 
detect  the  presence  of  as  low  a  content  as  5  per  cent  of  carbon 
monoxide  hemoglobin. 

4.  Neutral  Methaemoglobin. — Dilute  a  little  defibrinated  blood 
(1  :  10)  and  add  a  few  drops  of  a  freshly  prepared  10  per  cent 
solution  of  potassium  ferricyanide.  Shake  this  mixture  and  ob- 
serve that  the  bright  red  color  of  the  blood  is  displaced  by  a  brown- 
ish red.  Now  dilute  a  little  of  this  solution  and  examine  it  spec- 
troscopically.     Note   the   single,   very   dark   absorption-band   lying 

1  The  so-called  water  gas  with  which  ordinary  illuminating  gas  in  diluted  con- 
tains usually  as  much  as  20  per  cent  of  carbon  monoxide   (CO). 

2  This  transforms  the  oxyhemoglobin  into  methremoglobin. 

8  This  is  done  to   free  the  blood   from  carbon  monoxide  haemoglobin. 


206  PHYSIOLOGICAL    CHEMISTRY. 

to  the  left  of  D  and,  if  the  dilution  is  sufficiently  great,  also  ob- 
serve the  two  rather  faint  bands  lying  between  D  and  E  in  some- 
what similar  positions  to  those  occupied  by  the  absorption  bands 
of  oxyhemoglobin.  Add  a  few  drops  of  Stokes'  reagent  to  the 
methsemoglobin  solution  while  it  is  in  position  before  the  spectro- 
scope and  note  the  immediate  appearance  of  the  oxyhemoglobin 
spectrum  which  is  quickly  followed  by  that  of  hemoglobin. 

5.  Alkaline  Methaemoglobin. — Render  a  neutral  solution  of 
methsemoglobin,  such  as  that  used  in  the  last  experiment  (4), 
slightly  alkaline  with  a  few  drops  of  ammonia.  The  solution  be- 
comes redder  in  color,  due  to  the  formation  of  alkaline  methemo- 
globin  and  shows  a  spectrum  different  from  that  of  the  neutral 
body.  In  this  case  we  have  a  band  on  either  side  of  D,  the  one 
nearer  the  red  end  of  the  spectrum  being  much  the  fainter.  A 
third  band,  darker  than  either  of  those  mentioned,  lies  between  D 
and  E  somewhat  nearer  E. 

6.  Alkali  Haematin. — Observe  the  spectrum  of  the  alkali  he- 
matin  prepared  in  Experiment  16  on  page  199.  Also  make  a 
spectroscopic  examination  of  a  freshly  prepared  alkali  hematin.1 
The  typical  spectrum  of  alkali  hematin  shows  a  single  absorption- 
band  lying  across  D  and  mainly  toward  the  red  end  of  the  spectrum. 

7.  Reduced  Alkali  Haematin  or  Haemochromogen. — Dilute 
the  alkali  hematin  solution  used  in  the  last  experiment  (6)  to 
such  an  extent  that  it  shows  no  absorption  band.  Now  add  a  few 
drops  of  Stokes'  reagent  and  note  that  the  greenish-brown  color 
of  the  alkali  hematin  solution  is  displaced  by  a  bright  red  color. 
This  is  due  to  the  formation  of  hemochromogen  or  reduced  alkali 
hematin.  Examine  this  solution  spectroscopically  and  observe  the 
narrow,  dark  absorption-band  lying  midway  between  D  and  E.  If 
the  dilution  is  not  too  great  a  faint  band  may  be  observed  in  the 
green  extending  across  E  and  b. 

8.  Acid  Haematin. — To  some  defibrinated  blood  add  half  its  vol- 
ume of  glacial  acetic  acid  and  an  equal  volume  of  ether.  Mix 
thoroughly.  The  acidified  ethereal  solution  of  hematin  rises  to  the 
top  and  may  be  poured  off  and  used  for  the  spectroscopic  exam- 
ination. If  desired  it  may  be  diluted  with  acidified  ether  in  the 
ratio  of  one  part  of  glacial  acetic  acid  to  two  parts  of  ether.  A 
distinct  absorption-band  will  be  noted  in  the  red  between  C  and 

1  Alkali  haematin  may  be  prepared  by  mixing  one  volume  of  a  concentrated 
potassium  hydroxide  or  sodium  hydroxide  solution  and  two  volumes  of  dilute 
(1:5)  defibrinated  blood.  This  mixture  should  be  heated  gradually  almost  to 
boiling,  then  cooled  and  shaken  for  a  few  moments  in  the  air  before  examination. 


BLOOD.  207 

D  and  lying  somewhat  nearer  C  than  the  band  in  the  methaemo- 

globin  spectrum.  Between  J)  and  F  may  be  seen  a  rather  indis- 
tinct broad  band.  Dilute  the  solution  until  this  band  resolves  itself 
into  two  bands.  Of  these  the  more  prominent  is  a  broad,  dark 
absorption-band  lying  in  the  green  between  b  and  F.  The  second, 
a  narrow  band  of  faint  outline,  lies  in  the  light  green  to  the  red 
side  of  E.  A  fourth  very  faint  band  may  be  observed  lying  on 
the  violet  side  of  D. 

9.  Acid  Haematoporphyrin. — To  5  c.c.  of  concentrated  sul- 
phuric acid  in  a  test-tube  add  two  drops  of  blood,  mixing  thoroughly 
by  agitation  after  the  addition  of  each  drop.  A  wine-red  solution 
is  produced.  Examine  this  solution  spectroscopically.  Acid  hae- 
matoporphyrin gives  a  spectrum  with  an  absorption-band  on  either 
side  of  D,  the  one  nearer  the  red  end  of  the  spectrum  being  the 
narrower. 

10.  Alkaline  Haematoporphyrin. — Introduce  the  acid  haemato- 
porphyrin solution  just  examined  into  an  excess  of  distilled  water. 
Cool  the  solution  and  add  potassium  hydroxide  slowly  until  the 
reaction  is  but  slightly  acid.  A  colored  precipitate  forms  which 
includes  the  principal  portion  of  the  haematoporphyrin.  The 
presence  of  sodium  acetate  facilitates  the  formation  of  this  precipi- 
tate. Filter  off  the  precipitate  and  dissolve  it  in  a  small  amount  of 
dilute  potassium  hydroxide.  Alkaline  haematoporphyrin  prepared  in 
this  way  forms  a  bright  red  solution  and  possesses  four  absorption- 
bands.  The  first  is  a  very  faint,  narrow  band  in  the  red.  midway 
between  C  and  D ;  the  second  is  a  broader,  darker  band  lying  across 
D,  principally  to  the  violet  side.  The  third  absorption-band  lies 
principally  between  D  and  E,  extending  for  a  short  distance 
across  E  to  the  violet  side,  and  the  fourth  band  is  broad  and 
dark  and  lies  between  b  and  F.  The  first  band  mentioned  is  the 
faintest  of  the  four  and  is  the  first  to  disappear  when  the  solution 
is  diluted. 

VII.  Instruments  Used  in  the  Clinical  Examination  of  the  Blood. 

i.  Fleischl's  Haemometer  (Fig.  64,  p.  208). — This  is  an  instru- 
ment used  quite  extensively  clinically,  for  the  quantitative  deter- 
mination of  haemoglobin.  The  instrument  consists  of  a  small  cyl- 
inder which  is  provided  with  a  fixed  glass  bottom  and  a  movable 
glass  cover,  and  which  is  divided,  by  means  of  a  metal  septum,  into 
two  compartments  of  equal  capacity.  This  cylinder  is  supported 
in  a  vertical  position  by  means  of  a  mechanism  which  resembles 


208 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  64. 


Fleischl's  H^emometer. 
(Da  Costa.) 


the  base  and  stage  of  an  ordinary  microscope.  Underneath  the 
stage  is  placed  a  colored  glass  wedge  (see  Fig.  66,  p.  209),  so 
arranged  as  to  run  immediately  beneath  the  glass  bottom  of  one  of 
the  compartments  of  the  cylinder  and  ground  in  such  a  manner  that 

each  part  of  the  wedge  corresponds  in 
color  to  a  solution  of  haemoglobin  of 
some  definite  percentage.  The  glass 
wedge  is  held  in  a  metal  frame  and 
may  be  moved  backward  or  forward 
by  means  of  a  rack  and  pinion  ar- 
rangement. A  scale  along  the  side  of 
this  frame  indicates  the  percentage 
of  the  normal  amount  of  haemoglobin 
which  each  particular  variation  in  the 
depth  of  color  of  the  ground  wedge 
represents,  taking  the  normal  haemo- 
globin content  as  100.1  In  a  position 
corresponding  to  the  position  of  the 
mirror  on  the  ordinary  microscope  is  attached  a  light-colored  opaque 
plate  which  serves  to  reflect  the  light  upward  through  the  colored 
wedge  and  the  cylinder  to  the  eye  of  the  observer. 

In  making  a  determination  of  the  percentage  of  haemoglobin 
by  this  instrument  the  procedure  is  as  follows :  Fill  each  compart- 
ment about  three-fourths  full  of  distilled  water.  Puncture  the 
finger-tip  or  lobe  of  the  ear  of  the  subject  by 
means  of  a  sterile  needle  or  scalpel  and,  as  soon 
as  a  drop  of  blood  appears,  place  one  end  of 
the  capillary  pipette  (Fig.  65),  which  accom- 
panies the  instrument,  against  the  drop  and 
allow  it  to  fill  by  capillary  attraction.  To  prevent 
the  blood  from  adhering  to  the  exterior  of  the 
tube,  and  so  render  the  determination  inaccu- 
rate, it  is  customary  to  apply  a  very  thin  coat- 
ing of  mutton  fat  to  the  outer  surface  before 

using  or  to  wrap  the  tube  in  a  piece  of  oily  chamois  when  not  in 
use.  As  soon  as  the  tube  has  been  accurately  filled  with  blood 
it  should  be  dipped  into  the  water  of  one  of  the  compartments  of 
the  cylinder  and  all  traces  of  the  blood  washed  out  with  water  by 
means  of  a  small  dropper  which  accompanies  the  instrument.  If 
the  blood  is  not  well  distributed  throughout  the  compartment  and 


Fig.  65. 


Pipette  of  Fleischl's 
h^emometer. 


x  The  scale  of  the  ordinary  instrument  is  usually  too  high. 


BLOOD.  209 

does  not  form  a  homogeneous  solution  the  contents  of  the  com- 
partment should  be  mixed  thoroughly  by  means  of  the  metal  handle 
of  the  capillary  measuring  pipette.  When  this  has  been  done  each 
compartment  should  be  completely  filled  with  distilled  water  and 
the  glass  cover  adjusted,  care  being  taken  that  the  contents  of 
the  two  compartments  do  not  mix.  Now  adjust  the  cylinder  so 
that  the  compartment  containing  the  pure  distilled  water  is  im- 
mediately above  the  colored  glass 
wedge.  By  means  of  the  rack 
and  pinion  arrangement  manipu- 
late the  colored  wedge  until  a 
portion  of  it  is  found  which  cor- 
responds in  color  with  the  diluted 
blood.     When  this  agreement  in 

,         .  ,  ,    ,,  .    ,     Colored    Glass    Wedge    of    Fleischl's 

color  has  been  secured  the  point  j^mometer.    (Da  Costa.) 

on  the  scale  corresponding  to  this 

particular  color  should  be  read  and  the  actual  percentage  of  haemo- 
globin computed.  For  instance,  if  the  scale  reading  is  90  it  means 
that  the  blood  under  examination  contains  90  per  cent  of  the  normal 
quantity  of  haemoglobin,  i.  e.,  90  per  cent  of  14  per  cent. 

2.  Fleischl-Miescher  Haemometer. — The  apparatus  of  Fleischl 
has  recently  been  modified  by  Miescher.  If  all  precautions  are 
taken,  the  margin  of  error  in  the  absolute  quantity  of  haemoglobin 
determined  by  this  instrument  does  not  exceed  0.15-0.22  per  cent 
by  weight  of  the  blood.  Detailed  directions  for  the  manipulation 
of  the  Fleischl-Miescher  haemometer  accompany  the  instrument. 
In  brief  Miescher  modified  the  instrument  as  follows :  ( 1 )  The 
scale  of  each  instrument  is  supplied  with  a  caliber  table  of  absolute 
haemoglobin  values,  expressed  in  milligrams :  the  scale  of  Fleischl's 
haemometer  shows  the  percentage  of  haemoglobin  in  relation  to  an 
average  selected  somewhat  arbitrarily.  Thus  many  errors  arising 
from  the  irregular  coloring  of  the  glass  wedge  of  the  older  appara- 
tus are  avoided  in  the  instrument  as  modified.  (2)  Each  in- 
strument is  accompanied  by  a  measuring  pipette  (melangeur)  which 
allows  of  a  more  accurate  measurement  of  the  blood  than  was  pos- 
sible with  the  capillary  tubes  of  the  older  apparatus.  (3)  With 
the  aid  of  the  measuring  pipette  mentioned  above  blood  of  varying 
degrees  of  concentration  may  be  compared.  In  this  way  the  in- 
dividual examinations  are  controlled  and  a  check  upon  the  ac- 
curacy of  the  graduation  in  the  color  of  the  glass  wedge  is  also 
afforded.     This  wedge  is  much  more  evenly  and  accurately  colored 

15 


2IO 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  67. 


than  in  the  unmodified  apparatus  of  Fleischl.  (4)  Before  reading 
the  percentage  as  indicated  by  the  scale,  the  chamber  is  covered 
with  a  glass  and  a  diaphragm  which  sharply  define  the  field  on 
all  sides  without  the  formation  of  a  meniscus. 

The  measuring  pipette  is  constructed  essentially  the  same  as  the 
pipettes  which  accompany  the  Thoma-Zeiss  apparatus  (see  page  214) . 
The  capillary  portion,  however,  is  graduated  1,  2/z  and  ^4  which 
enables  the  observer  to  dilute  the  blood  sample  in  the  proportion 
of  1  :  200,  1  :  300  or  1  :  400  as  he  may  desire.  If  there  is  diffi- 
culty in  drawing  in  the  blood  exactly  to  one  of  the  graduations 
just  mentioned  the  amount  of  blood  above  or  below  the  volume  in- 
dicated by. the  graduation  may  be  determined  by  means  of  certain 

delicate  cross-lines  which  are 
placed  directly  above  and 
below  the  graduation.  Each 
cross-line  corresponds  to  %oo 
of  the  volume  of  the  capillary 
tube    from   the   tip  to  the    1 


graduation. 

A  0.1  per  cent  solution  of 
sodium  carbonate  is  used  to 
dissolve  the  stroma  of  the 
erythrocytes  and  so  render  the 
blood  solution  perfectly  clear. 
If  this  is  not  done  the  color 
of  the  blood  solution  invari- 
ably appears  darker  in  tone 
than  that  of  the  colored  glass 
wedge.  A  freshly  prepared 
sodium  carbonate  solution 
should  be  used  in  order  that 
the  clearness  of  the  solution 


Dare's  H^moglobinometer.     (Da  Costa.) 

R,  Milled  wheel  acting  by  a  friction 
bearing  on  the  rim  of  the  color  disc; 
S,  case  inclosing  color  disc,  and  provided 
with  a  stage  to  which  the  blood  cham- 
ber is  fitted ;  T,  movable  wing  which 
is  swung  outward  during  the  observation, 
to  serve  as  a  screen  for  the  observer's  eyes, 
and  which  acts  as  a  cover  to  inclose  the 
color  disc  when  the  instrument  is  not  in 
use;  U,  telescoping  camera  tube,  in  posi- 
tion for  examination ;  V,  aperture  admitting  may  not  be  marred  by  the 
light  for  illumination  of  the  color  disc;  X. 
capillary  blood  chamber  adjusted  to  stage 
of  instrument,  the  slip  of  opaque  glass,  W, 
being  nearest  to  the  source  of  light ;  Y, 
detachable  candle-holder  ;  Z,  rectangular  slot 
through  which  the  hemoglobin  scale  indi- 
cated on  the  rim  of  the  color  disc  is  read. 


presence  of   sodium   bicarbo- 
nate. 

3.     Dare's    Haemoglobin- 
ometer     (Fig.     67).  —  This 
instrument,  as  the  name  sig- 
nifies,  is  used   for  the  determination  of   haemoglobin.      In   using 
either   Fleischl's    haemometer   or   the    instrument    as   modified   by 
Miescher  the  blood  is  diluted  for  examination  whereas  with  the 


BLOOD. 


21  I 


Dare  instrument  no  dilution  is  required.  This  probably  allows  of 
rather  more  accurate  determinations  than  are  possible  with  the  old 
Fleischl  apparatus. 

The  instrument  consists  essentially  of  the  following'  parts :  (  \ ) 
A  capillary  observation  cell,  (2)  a  semicircular  colored  glass  wedge, 
(3)  a  milled  wheel  for  manipulating  the  wedge,  (4)  a  candle  used 
to  illuminate  portions  of  the  capillary  observation  cell  and  the 
colored  wedge,  (5)  a  small  telescope  used  in  the  examination  of 
the  areas  illuminated  by  the  candle  flame,  (6)  a  scale  graduated  in 
percentages  of  the  normal  amount  of  haemoglobin,  (7)  a  hard 
rubber  case,   (8)  a  movable  screen  attached  to  the  case. 

The  capillary  observation  cell  is  formed  of  two  small,  polished 
rectangular  plates  of  glass,  one  being  transparent  and  the  other 
opaque.  When  held  in  position  on  the  instrument,  by  means  of  a 
small  metal  bracket,  the  opaque  portion  of  the  cell  is  nearer  the 
candle  and  thus  serves  to  soften  the  glare  of  light  when  an  obser- 
vation is  being  made.  The  transparent  portion  of  the  cell  is  directly 
over  a  circular  opening  in  the  case,  through  which  the  blood  speci- 
men is  viewed  by  means  of  the  small  telescope. 

The  semicircular  colored  glass  wedge  is  so  ground  that  each 
particular  shade  of  color  corresponds  to  that  possessed  by  fresh 
blood  which  contains  some  definite  per- 
centage of  haemoglobin.  It  is  mounted 
upon  a  disc  which  may  be  manipulated 
by  the  milled  wheel  in  such  a  manner  as 
to  bring  successive  portions  of  the  wedge 
in  position  to  be  viewed  through  a  cir- 
cular opening  contiguous  to  the  opening- 
through  which  the  blood  specimen  is 
viewed.  For  a  further  description  of  the 
instrument  see  Figures  6j,  68  and  69,  on 
pages  210,  211  and  212,  respectively. 

In  using  the  Dare  haemoglobinometer 
proceed  as  follows :  Puncture  the  finger- 
tip or  lobe  of  the  ear  of  the  subject  by 

means  of  a  needle  or  scalpel  and,  after  a     Horizontal  Section-  of  Dare's 

..  HAEMOGLOBINOMETER.  * 

drop  of  blood  of  good  proportions  has  {Da  Costa.) 

formed,  place  the  flat  capillary  observa- 
tion cell  in  contact  with  the  drop  and  allow  it  to  fill  by  capillary 
attraction  (Fig.  69,  page  212).     Replace  the  cell  in  ils  proper  place 
on  the  instrument.     When  in  position,  a  portion  of  this  cell  may  be 


212  PHYSIOLOGICAL    CHEMISTRY. 

observed  through  a  small  telescope  attached  to  the  apparatus.  It  is 
viewed  through  a  circular  opening  and  near  this  circle  is  a  second 
one  through  which  a  portion  of  a  semicircular  colored  glass  wedge 
is  visible.  These  two  circles  are  illuminated  simultaneously  by 
means  of  the  flame  of  a  candle.  The  colored  glass  may  be  rotated 
by  means  of  a  milled  wheel  and  the  point  of  agreement  of  the  color 


Method  of  Filling  the  Capillary  Observation   Cell  of  Dare's 

H^MOGLOBINOMETER.        (Da    Costd.) 

of  the  adjoining  discs  may  be  determined  in  the  same  way  as  in 
Fleischl's  haemometer.  The  scale  reading  gives  the  percentage  of 
the  normal  quantity  of  haemoglobin  which  the  blood  sample  under 
examination  contains.  Compute  the  actual  haemoglobin  content  in 
the  same  manner  as  from  the  scale  reading  of  the  Fleischl  haemom- 
eter  (see  page  209). 

4.  Tallquist's  Haemoglobin  Scale. — This  consists  essentially  of 
a  series  of  ten  colors  corresponding  to  stains  produced  by  blood 
containing  varying  percentages  of  haemoglobin.  In  using  this  scale 
a  drop  of  blood  is  allowed  to  fall  on  a  small  section  of  filter  paper 
and  the  resulting  color  is  compared  with  the  ten  colors  of  the  scale. 
When  the  color  in  the  scale  is  found  which  corresponds  to  the  color 
of  the  blood  stain  the  accompanying  haemoglobin  value  is  read  off 
directly.  This  is  a  very  convenient  method  for  determining  haemo- 
globin at  the  bedside.  There  is  a  possibility  of  the  colors  being 
inaccurately  printed,  however,  and  even  if  originally  correct  in  tint, 
under  the  continued  influence  of  air  and  light  they  must  eventually 
alter  somewhat. 

5.  Thoma-Zeiss  Hasmocytometer.  —  This  is  an  instrument 
used  in  "blood  counting,"  i.  e.,  in  determining  the  number  of 
erythrocytes  and  leucocytes.  The  instrument  consists  of  a  micro- 
scopic slide  constructed  of  heavy  glass  and  provided  with  a  central 
counting  cell  (see  Fig.  70,  p.  213).    This  cell,  with  the  cover-glass 


BLOOD. 


213 


in  position,  is  exactly  o.  1  millimeter  deep.  The  floor  of  the  cell 
is  divided  by  delicate  lines  into  squares  each  of  which  is  %oo  of  a 
square  millimeter  in  area  (see  Fig.  72,  page  215).  The  volume  of 
blood  therefore  between  any  particular  square  and  the  cover-glass 
above  must  be  %ooo  cubic  millimeter.     Accompanying  each  instru- 

Fig.  70. 


Thoma-Zeiss    Counting   Chamber.      (Da   Costa.) 


ment  are  two  capillary  pipettes  (Fig.  71,  page  214),  each  constructed 
with  a  mixing  bulb  in  its  upper  portion.  Each  bulb  is  further  pro- 
vided with  an  enclosed  glass  bead  which  is  of  great  assistance  in 
mixing  the  contents  of  the  chamber.  The  stem  of  each  pipette  is 
graduated  in  tenths  from  the  tip  to  the  bulb.  The  final  graduation 
at  the  upper  end  of  the  bulb  is  101  on  the  pipette  used  in  mixing  the 
blood  sample  in  which  the  erythrocytes  are  counted  (erythrocy- 
tometer,  see  Fig.  71,  page  214),  and  11  on  the  pipette  used  in  mix- 
ing the  blood  sample  for  the  leucocyte  count  (leucocytometer,  see 
Fig.  71,  page  214).  In  making  "blood  counts"  with  the  hsemo- 
cytometer  it  is  necessary  to  use  some  diluting  fluid.  Two  very 
satisfactory  forms  of  fluid  for  this  purpose  are  Toison's  and  Sher- 
rington's solutions.1  When  either  of  these  solutions  is  used  as  the 
diluting  fluid  it  is  possible  to  make  a  very  satisfactory  count  of 
both  the  erythrocytes  and  leucocytes  from  the  same  preparation, 
since  the  leucocytes  are  stained  by  the  methyl-violet  or  methylene- 
blue. 

vIn  counting  the  erythrocytes  by  means  of  the  hsemocytometer, 
proceed  as  follows :     Thoroughly  cleanse  the  tip  of  the  finger  or 


1  Toison's    solution    has    the    follow- 
ing  formula : 

Methyl    violet 0.025  gram. 

Sodium    chloride I  gram. 

Sodium    sulphate 8  grams. 

Glycerol    30  grams. 

Distilled  water   160  grams. 


Sherrington's    solution    has   the    fol- 
lowing formula  : 

Methylene-blue     0.1  gram. 

Sodium    chloride 1.2  gram. 

Neutral      potassium      ox- 
alate          1.2  gram. 

Distilled    water 300.0  grams. 


214 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  71. 


lobe  of  the  ear  of  the  subject  by  the  use  of  soap  and  water,  alcohol 
and  ether  applied  in  the  sequence  just  given.  Puncture  the  skin 
by  means  of  a  needle  or  scalpel  and  allow  the  blood  drop  to  form 
without  pressure.  Place  the  tip  of  the  pipette  in  contact  with  the 
blood  drop,  being  careful  to  avoid  touching  the  skin,  and  draw 
blood  into  the  pipette  up  to  •the  point  marked  0.5  or  1  according 
to  the  desired  dilution.  Rapidly  wipe  the  tip  of 
the  pipette  and  immediately  fill  it  to  the  point 
marked  101  with  Toison's  or  Sherrington's  solu- 
tion. Now  thoroughly  mix  the  blood  and  dilut- 
ing fluid  within  the  mixing  chamber  by  tapping 
the  pipette  gently  against  the  finger,  or  by  shaking 
it  while  held  securely  with  the  thumb  at  one  end 
and  the  middle  finger  at  the  other.  After  the  two 
fluids  have  been  thoroughly  mixed  the  diluting 
fluid  contained  in  the  capillary-tube  below  the 
bulb  should  be  discarded  in  order  to  insure  the 
collection  of  a  drop  of  the  thoroughly  mixed  blood 
and  diluting  solution  for  examination.  Transfer 
a  drop  from  the  pipette  to  the  ruled  floor  of  the 
counting  chamber  and,  after  placing  the  cover- 
glass  firmly  in  position,1  allow  an  interval  of  a 
few  minutes  to  elapse  for  the  corpuscles  to  settle 
before  making  the  count.  Now  place  the  slide 
under  the  microscope  and  count  the  number  of 
erythrocytes  in  a  number  of  squares,  counting  the 
corpuscles  which  are  in  contact  with  the  upper 
and  the  right-hand  boundaries  of  the  square  as 
belonging  to  that  square.  Take  the  squares  in 
some  definite  sequence  in  order  that  the  recount- 
ing of  the  same  corpuscles  may  be  avoided.  Of 
course,  all  things  being  equal,  the  greater  the 
number  of  squares  examined  the  more  accurate 
the  count.  It  is  considered  essential  under  all 
circumstances,  where  an  accurate  count  is  desired,' 
that  the  counting  chamber  shall  be  filled,  at  least  twice,  and  the  indi- 
vidual counts  made  in  each  instance,  as  indicated  above,  before  the 
data  are  deemed  satisfactory. 

1  If  the  cover  glass  is  in  accurate  apposition  to  the  counting  cell  Newton's 
rings  may  be  plainly  observed. 


Thoma-Zeiss     Cap- 
illary Pipettes. 

A,       Erythrocytom- 

eter ;  B,  Leuco- 

cytometer. 


BLOOD.  2 1  5 

To  calculate  the  number  of  erythrocytes  per  cubic  millimeter 
of  undiluted  blood  proceed  as  follows :  Determine  the  number  of 
corpuscles  in  any  given  number  of  squares  and  divide  this  total  by 
the   number   of   squares,   thus   obtaining   the   average   number   of 


Fig 


Ordinary  Ruling  of  Thoma-Zeiss  Counting  Chamber.      {Da  Costa.) 

erythrocytes  per  square.  Multiply  this  average  by  4,000  to  obtain 
the  number  of  erythrocytes  per  cubic  millimeter  of  diluted  blood, 
and  multiply  this  product  by  100  or  200,  according  to  the  dilution, 
to  obtain  the  number  of  erythrocytes  per  cubic  millimeter  of  undi- 
luted blood.     Thus : 

Average  number  of  erythro-  v,  v ,  ,  .       Number      of      erythrocytes 

X  4,ooo  X  200  (or  100)  =  .... 

cytes  per  square  per  cubic  millimeter. 

Great  care  should  be  taken  to  see  that  the  capillary  pipette  is 
properly  cleaned.  After  using,  it  should  be  immediately  rinsed  out 
with  the  diluting  fluid,  then  with  water,  alcohol  and  ether  in  the 
sequence  given.  Finally  dry  air  should  be  drawn  through  the 
capillary  and  a  horse  hair  inserted  to  prevent  the  entrance  of  dust 
particles. 

In  counting  leucocytes  by  means  of  the  haemocytometer  proceed 
as  follows:  As  mentioned  above,  if  the  diluting  fluid  is  either 
Toison's  or  Sherrington's  solution  the  leucocytes  may  be  counted 
in  the  same  specimen  of  blood  in  which  the  erythrocytes  are  counted. 
When  this  is  done  it  is  customary  to  use  a  slide  provided  with 
Zappert's  modified  ruling  (Fig.  73,  p.  216).    This  method  is  rather 


2l6  PHYSIOLOGICAL    CHEMISTRY. 

more  accurate  than  the  older  one  of  counting  the  leucocytes  in  a 
separate  specimen  of  blood.  Furthermore  it  is  obviously  preferable 
to  count  both  the  erythrocytes  and  the  leucocytes  from  the  same 
blood  sample.     To  insure  accuracy  the  number  of  leucocytes  within 

Fig.  73. 


Zappert's  Modified  Ruling  of  Thoma-Zeiss  Counting  Chamber.     (Da  Costa.) 

the  whole  ruled  region  should  be  determined  in  duplicate  blood 
samples.  This  includes  the  examination  of  an  area  eighteen  times 
as  great  as  the  old  style  Thoma-Zeiss  central  ruling.  This  region 
then  would  correspond  to  3,600  of  the  small  squares  and,  if  dupli- 
cate examinations  were  made,  the  total  number  of  small  squares 
examined  would  aggregate  7,200.  The  calculation  would  be  as 
follows : 

Number    of    leucocytes    in  K ,         ^y  Number    of    leucocytes    per 

X  200  X  4,000  -^  7,200  =         ,.         .„. 
7,200  squares  cubic  millimeter. 

If  a  Zappert  slide  is  not  available,  a  good  plan  to  follow  is  to 
place  a  diaphragm  in  the  tube  of  the  ocular  of  the  microscope  con- 
sisting of  a  circle  of  black  cardboard  or  metal1  having  a  square 
hole  in  the  center  of  such  a  size  as  to  allow  of  the  examination  of 
exactly  100  squares  or  one-fourth  of  a  square  millimeter  at  one 
time.  With  this  arrangement  any  portion  of  the  specimen  may 
be  examined  and  counted  whether  within  or  without  the  ruled  area. 
In  counting  by  means  of  this  device  it  is,  of  course,  helpful  if  the 

^hrlich's  mechanical  eye-piece  with  iris  diaphragm  is  also  very  satisfactory 
for  this  purpose. 


BLOOD.  2  I  / 

microscope  is  provided  with  a  mechanical  stage,  but  even  without 
this  arrangement,  if  the  observer  is  careful  to  see  that  the  leuco- 
cytes at  the  extreme  boundary  of  one  field  move  to  the  opposite 
boundary  when  the  position  of  the  slide  is  changed,  the  device 
may  be  very  satisfactorily  employed.  The  leucocytes  should  be 
counted  in  36  of  the  diaphragm-fields  in  duplicate  specimens  and 
the  calculation  made  in  the  same  manner  as  explained  above. 

If  the  leucocytes  are  counted  in  a  separate  specimen  of  blood 
ordinarily  the  diluting  fluid  is  0.3-0.5  per  cent  acetic  acid,  a  fluid 
in  which  the  leucocytes  alone  remain  visible.  Under  these  conditions 
the  dilution  is  customarily  made  in  the  pipette  having  11  as  the 
final  graduation.  The  capillary  portion  is  of  larger  caliber  and 
so  requires  a  greater  amount  of  blood  to  fill  it  to  the  0.5  or  1 
mark  than  is  required  in  the  use  of  the  other  form  of  pipette.  In 
counting  the  leucocytes  according  to  this  method  it  is  customary 
to  draw  blood  into  the  pipette  up  to  the  1  mark  and  immediately 
fill  the  remaining  portion  of  the  apparatus  to  the  1 1  graduation 
with  the  0.3-0.5  per  cent  acetic  acid.  It  then  remains  to  count  the 
number  of  leucocytes  in  the  whole  central  ruled  portion  of  400 
squares.  This  should  be  done  in  duplicate  samples  and  the  calcula- 
tion made  as  follows : 

Number    of    leucocytes    in  .,  ■-.  0  Number    of    leucocytes   per 

J  X  4,000  X 10  -7-  800    =  ,.        .... 

800  squares.  cubic  millimeter. 


CHAPTER   XIII. 

MILK. 

Milk  is  the  most  satisfactory  individual  food  material  elaborated 
by  nature.  It  contains  the  three  nutrients,  protein,  fat  and  carbo- 
hydrate and  inorganic  salts  in  such  proportion  as  to  render  it  a 
very  acceptable  dietary  constituent.  It  is  a  specific  product  of 
the  secretory  activity  of  the  mammary  gland.  It  contains,  as  the 
principal  solids,  tri-olein,  tri-palmitin,  tri-stearin,  tri-butyrin,  case- 
inogen, lactalbumin,  lacto- globulin,  lactose  and  calcium  phosphate. 
It  also  contains  at  least  traces  of  lecithin,  cholesterol,  urea,  creatine, 
creatinine  and  the  tri-glycerides  of  caproic,  lauric  and  myristic  acids. 
Citric  acid  is  also  said  to  be  present  in  milk  in  minute  quantity. 
Fresh  milk  is  amphoteric  in  reaction  to  litmus,1  but  upon  standing 
for  a  sufficiently  long  time,  unsterilized,  it  becomes  acid  in  reaction, 
due  to  the  production  of  fermentation  lactic  acid, 

H    OH 

H-C-C-COOH, 

I      I 
H    H 

from  the  lactose  contained  in  it.  This  is  brought  about  through 
bacterial  activity.  The  white  color  is  imparted  to  the  milk  partly 
through  the  fine  emulsion  of  the  fat  and  partly  through  the  medium 
of  the  caseinogen  in  solution.  The  specific  gravity  of  milk  varies 
somewhat,  the  average  being  about  1.030.  Its  freezing-point  is 
about  — 0.560  C. 

Fresh  milk  does  not  coagulate  on  being  boiled  but  a  film  con- 
sisting of  a  combination  of  caseinogen  forms  on  the  surface.  If 
the  film  be  removed,  thus  allowing  a  fresh  surface  to  come  in 
contact  with  the  air,  a  new  film  will  form  indefinitely  upon  the 
application  of  heat.  Surface  evaporation  and  the  presence  of  fat 
facilitate  the  formation  of  the  film  but  are  not  essential  (Rettger). 
As  Jamison  and  Hertz  have  shown,  a  similar  film  will  form  on 
heating  any  protein  solution  containing  fat  or  paraffin.     If  the 

1  Human  milk  as  well  as  cow's  milk.     It  is,  however,  acid  to  phenolphthalein. 

218 


MILK.  219 

milk  is  acid  in  reaction,  through  the  inception  of  lactic  acid  fer- 
mentation, or  from  any  other  cause,  no  film  will  form  when  heat 
is  applied,  but  instead  a  true  coagulation  will  occur.  When  milk 
is  boiled  certain  changes  occur  in  its  odor  and  taste.  These  changes, 
according  to  Rettger,  are  due  to  a  partial  decomposition  of  the  milk 
proteins  and  are  accompanied  by  the  liberation  of  a  volatile  sul- 
phide, probably  hydrogen  sulphide. 

The  milk-curdling  enzymes  of  the  gastric  and  the  pancreatic 
juice  have  the  power  of  splitting  the  caseinogen  of  the  milk, 
through  a  process  of  hydrolysis,  into  soluble  casein  and  a  peptone- 

Fig.  74. 


■"       ■     ^%- 


C' 


J 


- 


f  < 


b    ■ 


Normal  Milk  and  Colostrum. 
a,  Normal  milk  ;  b,  Colostrum. 

like  body.  This  soluble  casein  then  forms  a  combination  with  the 
calcium  of  the  milk  and  an  insoluble  curd  of  calcium  casein  or 
casein  results.  The  clear  fluid  surrounding  the  curd  is  known 
as  whey. 

The  most  pronounced  difference  between  human  milk  and  cow's 
milk  is  in  the  protein  content,  although  there  are  also  differences 
in  the  fats  and  likewise  striking  biological  differences  difficult  to 
define  chemically.  It  has  been  shown  that  the  caseinogen  of  human 
milk  differs  from  the  caseinogen  of  cow's  milk  in  being  more  diffi- 
cult to  precipitate  by  acid  or  coagulate  by  gastric  rennin.  The 
casein  curd  also  forms  in  a  much  looser  and  more  flocculent  manner 
than  that  from  cow's  milk  and  is  for  this  reason  much  more  easily 
digested  than  the  latter.  Interesting  data  relative  to  the  composi- 
tion of  milk  from  various  sources,  may  be  gathered  from  the  fol- 


220 


PHYSIOLOGICAL    CHEMISTRY. 


lowing  table  which  was  compiled  mainly  from  the  results  of  inves- 
tigations by  Bunge  and  by  Abderhalden.  It  will  be  noted  that  the 
composition  of  the  milk  varies  directly  with  the  length  of  time 
needed  for  the  young  of  the  particular  species  to  double  in  weight. 


Period  in  Which 
Weight  of  the 
New-born  is 

Doubled  (Days). 

100  Parts  of  Milk  Contain 

Species. 

| 
Proteins.                    Salts. 

Calcium. 

Phosphoric 
Acid. 

Man 

1 80 

60 

47 

22 

*5 
14 

9-5 

9 

6 

1.6 
2.0 
3-5 

3-7 
4.9 

0.2 
0.4 
0.7 
0.8 
0.8 

O.O33 
O.I24 
O.160 
O.I97 
O.245 
O.249 

o-455 
0.891 

O.O47 

Horse 

0.I3I  ' 

O.I97 

O.284 

O.293 

O.308 

O.508 
O.997 

Pig 

5.2                   0.8 

Cat 

Dog 

Rabbit 

7.0 

7-4 
10.4 

1.0 

2-5 

Lactose,  the  principal  carbohydrate  constituent  of  milk,   is  an 
important  member  of  the  disaccharide  group.     It  occurs  only  in 


Fig.    75. 


Lactose. 


milk,  except  as  it  is  found  in  the  urine  of  women  during  preg- 
nancy, during  the  nursing  period  and  soon  after  weaning;  it  also 
occurs  in  the  urine  of  normal  persons  after  the  ingestion  of  a  very 
large  amount  of  lactose  in  the  food.  It  is  not  derived  directly 
from  the  blood  but  is  a  specific  product  of  the  cellular  activity  of 
the  mammary  gland.  It  has  strong  reducing  power,  is  dextro- 
rotatory and  forms  an  osazone  with  phenylhydrazine.  The  souring 
of  milk  is  due  to  the  formation  of  lactic  acid  from  lactose  through 


MILK.  221 

the  agency  of  the  bacterium  lactis.  Putrefactive  bacteria  in  the 
alimentary  canal  may  bring  about  this  same  reaction.  Lactose  is 
not  fermentable  by  pure  yeast.  It  was  recently  claimed  that  lac- 
tosin,  a  new  carbohydrate,  had  been  isolated  from  milk. 

Caseinogen,  the  principal  protein  constituent  of  milk  belongs  to 
the  group  of  phosphoproteins.  It  has  acidic  properties  and  com- 
bines with  bases  to  produce  salts.  It  is  not  coagulable  upon  boiling 
and  is  precipitated  from  its  neutral  solution  by  certain  metallic  salts 
as  well  as  upon  saturation  with  sodium  chloride  or  magnesium  sul- 
phate.   Its  acid  solution  is  precipitated  by  an  excess  of  mineral  acid. 

Lactalbumin  and  lacto-globulin,  the  protein  constituents  of  milk, 
next  in  importance  to  caseinogen,  closely  resemble  serum  albumin 
and  serum  globulin  in  their  general  properties.  According  to  Wrob- 
lewski,  a  protein  called  opalisiu  is  also  present  in  milk. 

Colostrum  is  the  name  given  to  the  product  of  the  mammary- 
gland  secreted  for  a  short  time  before  parturition  and  during  the 
early  period  of  lactation  (see  Fig.  74,  p.  219).  It  is  yellowish  in 
color,  contains  more  solid  matter  than  ordinary  milk  and  has  a 
higher  specific  gravity  (1. 040-1. 080).  The  most  striking  differ- 
ence between  colostrum  and  ordinary  milk  is  the  high  percentage 
of  lactalbumin  and  lacto-globulin  in  the  former.  This  abnormality 
in  the  protein  content  is  responsible  for  the  coagulation  of  colos- 
trum upon  boiling. 

Such  enzymes  as  lipase,  amylase,  galactase,  catalase,  oxidases, 
peroxidases  and  reductases  have  been  identified  in  milk,  but  not 
all  of  them  in  milk  of  the  same  species  of  animal. 

Among  the  principal  preservatives  used  in  connection  with  milk 
are  formaldehyde,  hydrogen  peroxide,  boric  acid,  borates,  salicylic 
acid  and  salicylates. 

Experiments  on  Milk. 

1.  Reaction. — Test  the  reaction  of  fresh  cow's  milk  to  litmus. 

2.  Biuret  Test. — Make  the  biuret  test  according  to  directions 
given  on  page  92. 

3.  Microscopical  Examination. — Examine  fresh  zcJiole  milk, 
skimmed  or  centrifugated  milk  and  colostrum  under  the  microscope. 
Compare  the  microscopical  appearance  with  Fig.  74,  page  219. 

4.  Specific  Gravity.  —  Determine  the  specific  gravity  of  both 
whole  and  skimmed  milk  (see  p.  218).  Which  possesses  the  higher 
specific  gravity?     Explain  why  this  is  so. 

5.  Film  Formation. — Place   10  c.c.  of  milk  in  a  small  beaker 


222  PHYSIOLOGICAL    CHEMISTRY. 

and  boil  a  few  minutes.  Note  the  formation  of  a  film.  Remove 
the  film  and  heat  again.  Does  the  film  now  form?  Of  what  sub- 
stance is  this  film  composed  ?  The  biuret  test  was  positive,  why  do 
we  not  get  a  coagulation  here  when  we  heat  to  boiling? 

6.  Coagulation  Test. — Place  about  5  c.c.  of  milk  in  a  test-tube, 
acidify  slightly  with  dilute  acetic  acid  and  heat  to  boiling.  Do  you 
get  any  coagulation?     Why? 

7.  Action  of  Hot  Alkali. — To  a  little  milk  in  a  test-tube  add  a 
few  drops  of  potassium  hydroxide  and  heat.  A  yellow  color  de- 
velops and  gradually  deepens  into  a  brown.  To  what  is  the  forma- 
tion of  this  color  due? 

8.  Test  for  Chlorides. — To  about  5  c.c.  of  milk  in  a  test-tube 
add  a  few  drops  of  very  dilute  nitric  acid  to  form  a  precipitate. 
Filter  off  this  precipitate  and  test  the  filtrate  for  chlorides.  Does 
milk  contain  any  chlorides? 

9.  Guaiac  Test. — To  about  5  c.c.  of  water  in  a  test-tube  add  3 
drops  of  milk  and  enough  alcoholic  solution  of  guaiac  (strength 
about  i:6o)x  to  cause  a  turbidity.  Thoroughly  mix  the  fluids 
by  shaking  and  observe  any  change  which  may  gradually  take  place 
in  the  color  of  the  mixture.  If  no  blue  color  appears  in  a  short 
time,  heat  the  tube  gently  below  6o°  C.  and  observe  whether  the 
color  reaction  is  hastened.  In  case  a  blue  color  does  not  appear 
in  the  course  of  a  few  minutes,  add  hydrogen  peroxide  or  old 
turpentine,  drop  by  drop,  until  the  color  is  observed.  Fresh  milk 
will  frequently  give  this  blue  color  when  treated  with  an  alcoholic 
solution  of  guaiac  without  the  addition  of  hydrogen  peroxide  or  old 
turpentine.     See  discussion  on  page  192. 

10.  Kastle's  Peroxidase  Reaction. — The  peroxidase  reaction 
of  milk  is  founded  upon  the  fact  that  small  amounts  of  raw  milk 
will  induce  the  oxidation  of  various  leuco  compounds  by  hydrogen 
peroxide.  This  reaction  has  been  used  in  a  practical  way  as  the 
most  convenient  means  of  differentiating  between  raw  milk  and 
boiled  milk.  Many  substances  have  been  employed  for  this  purpose, 
e.  g.,  guaiac,  paraphenylenediamine,  ortol,  amidol,  etc.  Kastle  has 
found  that  a  dilute  solution  of  "trikresol"2  acts  as  a  sensitizing 
agent  in  the  peroxidase  reaction  and  offers  the  following  test  which 
is  based  upon  this  fact:  To  2-5  c.c.  of  raw  milk  in  a  test-tube  add 

1  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid 
instead  of  an  alcoholic  solution  of  guaiac  resin.  Guaiaconic  acid  is  a  constituent 
of  guaiac  resin. 

""Trikresol"  is  the  trade  .name  of  an  antiseptic  which  contains  the  three 
cresols  in  approximately  equal  proportions. 


MILK.  223 

0.1-0.3  c.c.  of  M/10  hydrogen  peroxide  and  1  c.c.  of  a  one  per  cent 
solution  of  "trikresol."  A  slight,  though  unmistakable  yellow 
color  will  be  observed  to  develop  throughout  the  solution. 

Repeat  the  test  using  milk  which  has  been  boiled  or  heated  to 
8o°  C.  for  10-20  minutes,  and  cooled,  and  note  that  no  yellow 
color  is  produced. 

The  color  reaction  in  the  case  of  the  raw  milk  probably  results 
from  the  oxidation  of  the  cresols  by  the  hydrogen  peroxide.  The 
first  product  of  this  oxidation1  then  oxidizes  the  leuco  compound, 
when  such  is  present,  and  causes  the  color  observed. 

11.  Saturation  with  Magnesium  Sulphate. — Place  about  5  c.c. 
of  milk  in  a  test-tube  and  saturate  with  solid  magnesium  sulphate. 
What  is  this  precipitate? 

12.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of 
five  tubes  as  follows : 

(a)  5  c.c.  of  fresh  milk  -J-  0.2  per  cent  HC1  (add  drop  by 
drop  until  a  precipitate  forms). 

(b)  5  c.c.  of  fresh  milk  -|-   5  drops  of  rennin  solution. 

(c)  5  c.c.  of  fresh  milk  -f-   10  drops  of  0.5  per  cent  Na2C03. 

(d)  5  c.c.  of  fresh  milk  -f-   10  drops  of  ammonium  oxalate. 

(e)  5  c.c.  of  fresh  milk  -f-   5  drops  of  0.2  per  cent  HC1. 
Now  to  each  of  the  tubes    (c),    (d)   and   (<?)   add  5  drops  of 

rennin  solution.  Place  the  whole  series  of  five  tubes  at  400  C. 
and  after  10-15  minutes  note  what  is  occurring  in  the  different 
tubes.     Give  a  reason  for  each  particular  result. 

13.  Preparation  of  Caseinogen. — Fill  a  large  beaker  one-third 
full  of  skimmed  (or  centrifugated)  milk  and  dilute  it  with  an  equal 
volume  of  water.  Add  dilute  hydrochloric  acid  until  a  flocculent 
precipitate  forms.  Stir  after  each  acidification  and  do  not  add  an 
excess  of  the  acid  as  the  precipitate  would  dissolve.  Allow  the 
precipitate  to  settle,  decant  the  supernatant  fluid  and  reserve  it  for 
use  in  later  (14-16)  experiments.  Filter  off  the  precipitate  of 
caseinogen  and  remove  the  excess  of  moisture  by  pressing  it  be- 
tween filter  papers.  Transfer  the  caseinogen  to  a  small  beaker,  add 
enough  95  per  cent  alcohol  to  cover  it  and  stir  for  a  few  moments. 
Filter,  and  press  the  precipitate  between  filter  papers  to  remove 
the  alcohol.  Transfer  the  caseinogen  again  to  a  small  dry  beaker, 
cover  the  precipitate  with  ether  and  heat  on  a  water-bath  for  ten 
minutes,  stirring  continuously.  Filter  (reserve  the  filtrate),  and 
press  the  precipitate  as  dry  as  possible  between  filter  papers.     Open 

1  Probably  some  organic  peroxide  or  quinoid  compound. 


224  PHYSIOLOGICAL    CHEMISTRY. 

the  papers  and  allow  the  ether  to  evaporate  spontaneously.  Grind 
the  precipitate  to  a  powder  in  a  mortar.  Upon  the  caseinogen  pre- 
pared in  this  way  make  the  following  tests : 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 

(b)  Millon's  Reaction. — Make  the  test  according  to  the  direc- 
tions given  on  page  90. 

(c)  Biuret  Test. — Make  the  test  according  to  directions  given  on 
page  92. 

(d)  Hopkins-Coie  Reaction. — Make  the  test  according  to  the  di- 
rections given  on  page  10 1. 

(e)  Loosely  Combined  Sulphur. — Test  for  loosely  combined  sul- 
phur according  to  the  directions  given  on  page  102. 

(/)  Fusion  Test  for  Phosphorus. — Test  for  phosphorus  by  fusion 
according  to  directions  given  on  page  251. 

14.  Coagulable  Proteins  of  Milk. — Place  the  filtrate  from  the 
original  caseinogen  precipitate  in  a  casserole  and  heat,  on  a  wire 
gauze,  over  a  free  flame.  As  the  solution  concentrates,  a  coagulum 
consisting  of  lactalbumin  and  lactoglobulin  will  form.  Continue 
to  concentrate  the  solution  until  the  volume  is  about  one-half  that 
of  the  original  solution.  Filter  off  the  coagulable  proteins  (re- 
serve the  filtrate)    and  test  them  as  follows : 

(a)  Millon's  Reaction. — Make  the  test  according  to  the  direc- 
tions given  on  page  90. 

(b)  Biuret  Test — Make  the  test  according  to  the  directions  given 
on  page  92. 

(c)  Hopkins-Cole  Reaction. — Make  the  test  according  .to  the  di- 
rections given  on  page  101. 

15.  Detection  of  Calcium  Phosphate. — Evaporate  the  filtrate 

from  the  coagulable  proteins,  on  a  water- 
bath,  until  crystals  begin  to  form.  It  may 
be  necessary  to  concentrate  to  15  c.c.  before 
any  crystallization  will  be  observed.  Cool 
the  solution,  filter  off  the  crystals  (reserve 
the  filtrate)  and  test  them  as  follows: 
(a)  Microscopical  Examination. — Exam- 

Cal'cium  PhoIphate.  ine  the  crystals  and  compare  them  with  those 
in  Fig.  j6. 

(b)  Dissolve  the  crystals  in  nitric  acid.  Test  part  of  the  acid 
solution  for  phosphates.  Render  the  remainder  of  the  solution 
slightly  alkaline  with  ammonia,  then  acidify  with  acetic  acid  and 
add  ammonium  oxalate.  Examine  the  crystals  under  the  micro- 
scope and  compare  them  with  those  in  Fig.  99,  p.  345. 


MILK.  225 

16.  Detection  of  Lactose. — Concentrate  the  filtrate  from  the 
calcium  phosphate  until  it  is  of  a  syrup-like  consistency.  Allow  it 
to  stand  over  night  and  observe  the  formation  of  crystals  of  lac- 
tose.    Make  the  following  experiments  : 

(a)  Microscopical  Examination. — Examine  the  crystals  and  com- 
pare them  with  those  in  Fig.  75,  page  220. 

(b)  Fehling's  Test. — Try  Fehling's  test  upon  the  mother  liquor. 

(c)  Phenylhydrazine  Test. — Apply  the  phenylhydrazine  test  to 
some  of  the  mother  liquor  according  to  the  directions  given  on 
page  24. 

17.  Milk  Fat. —  (a)  Evaporate  the  ether  filtrate  from  the  case- 
inogen  (Experiment  13)  and  observe  the  fatty  residue.  The  milk 
fat  was  carried  down  with  the  precipitate  of  caseinogen  and  was 
removed  when  the  latter  was  treated  with  ether.  If  centrifugated 
milk  was  used  in  the  preparation  of  the  caseinogen  the  amount 
of  fat  in  the  ether  filtrate  may  be  very  small.  To  secure  a  larger 
yield  of  fat  proceed  according  to  directions  given  under  (&)  below. 

(b)  To  25  c.c.  of  whole  milk  in  an  evaporating  dish  add  a 
little  sand  or  filter  paper  and  evaporate  the  fluid  to  dryness  on  a 
water-bath.  Grind  or  break  up  the  residue  after  cooling  and  ex- 
tract with  ether  in  a  flask.  Filter  and  remove  the  ether  from 
the  filtrate  by  evaporation.  How  can  you  identify  fats  in  the 
ethereal  residue? 

18.  Saponification  of  Butter. — Dissolve  a  small  amount  of  but- 
ter in  alcohol  made  strongly  alkaline  with  potassium  hydroxide. 
Place  the  alcoholic-potash  solution  in  a  casserole,  add  about  100 
c.c.  of  water  and  boil  for  10-15  minutes  or  until  the  odor  of  alco- 
hol cannot  be  detected.  Place  the  casserole  in  a  hood  and  neutralize 
the  solution  with  sulphuric  acid.  Note  the  odor  of  volatile  fatty 
acids,  particularly  butyric  acid. 

19.  Detection  of  Preservatives. —  (a)    Formaldehyde. 

I.  Gallic  Acid  Test. — Acidify  30  c.c.  of  milk  with  2  c.c.  of  nor- 
mal sulphuric  acid  and  distil.  Add  0.2-0.3  c.c.  of  a  saturated 
alcoholic  solution  of  gallic  acid  to  the  first  5  c.c.  of  the  distillate, 
then  incline  the  test-tube  and  slowly  introduce  3-5  c.c.  of  concen- 
trated sulphuric  acid,  allowing  it  to  run  slowly  down  the  side 
of  the  tube.  A  green  ring,  which  finally  changes  to  blue,  is  formed 
at  the  juncture  of  the  fluids.  This  is  claimed,  by  Sherman,  to  be 
twice  as  delicate  as  either  the  sulphuric  acid  or  the  hydrochloric 
acid  test  for  formaldehyde. 

II.  Leach's  Hydrochloric  Acid  Test. — Mix  10  c.c.  of  milk  and 
16 


226  PHYSIOLOGICAL    CHEMISTRY. 

io  c.c.  of  concentrated  hydrochloric  acid  containing  about  0.002 
gram  of  ferric  chloride  in  a  small  porcelain  evaporating  dish  or 
casserole  and  gradually  raise  the  temperature  of  the  mixture,  on 
a  water-bath,  nearly  to  the  boiling-point,  with  occasional  stirring. 
If  formaldehyde  is  present  a  violet  color  is  produced, while  a  brown 
color  develops  in  the  absence  of  formaldehyde.  In  case  of  doubt 
the  mixture,  after  having  been  heated  nearly  to  the  boiling-point  for 
about  one  minute,  should  be  diluted  with  50-75  c.c.  of  water,  and 
the  color  of  the  diluted  fluid  carefully  noted,  since  the  violet  color 
if  present  will  quickly  disappear.  Formaldehyde  may  be  detected 
by  this  test  when  present  in  the  proportion  1  :  250,000. 

(b)  Salicylic  and  Salicylates. — Remont's  Method.1  Acidify  20 
c.c.  of  milk  with  sulphuric  acid,  shake  well  to  break  up  the  curd, 
add  25  c.c.  of  ether,  mix  thoroughly  and  allow  the  mixture  to 
stand.  By  means  of  a  pipette  remove  5  c.c.  of  the  ethereal  extract, 
evaporate  it  to  dryness,  boil  the  residue  with  10  c.c.  of  40  per  cent 
alcohol  and  cool  the  alcoholic  solution.  Make  the  volume  10  c.c, 
filter  through  a  dry  paper  if  necessary  to  remove  fat,  and  to  5  c.c. 
of  the  filtrate,  which  represents  2  c.c.  of  milk,  add  2  c.c.  of  a  2  per 
cent  solution  of  ferric  chloride.  The  production  of  a  purple  or 
violet  color  indicates  the  presence  of  salicylic  acid. 

This  test  may  form  the  basis  of  a  quantitative  method  by  dilut- 
ing the  final  solution  to  50  c.c.  and  comparing  this  with  standard 
solutions  of  salicylic  acid.  The  colorimetric  comparisons  may  be 
made  in  a  Duboscq  colorimeter. 

(c)  Hydrogen  Peroxide. — Add  2-3  drops  of  a  2  per  cent  aque- 
ous solution  of  para-phenylenediamine  hydrochloride  to  10-15  c-c- 
of  milk.  If  hydrogen  peroxide  is  present  a  blue  color  will  be  pro- 
duced immediately  upon  shaking  the  mixture  or  after  allowing  it 
to  stand  for  a  few  minutes.  It  is  claimed  that  hydrogen  peroxide 
may  be  detected  by  this  test  when  present  in  the  proportion 
1 :  40,000. 

(d)  Boric  Acid  and  Borates. — To  the  ash,  obtained  according 
to  the  directions  given  in  Experiment  4,  Chapter  XXIII,  add  2  drops 
of  dilute  hydrochloric  acid  and  1  c.c.  of  water.  Place  a  strip  of  tur- 
meric paper  in  the  dish  and  after  allowing  it  to  soak  for  about  one 
minute  remove  it  and  allow  it  to  dry  in  the  air.  The  presence  of  boric 
acid  is  indicated  by  the  production  of  a  deep  red  color  which  changes 
to  green  or  blue  upon  treatment  with  a  dilute  alkali.  This  test  is  sup- 
posed to  show  boric  acid  when  present  in  the  proportion  1 :  8000. 

1  Sherman's  Organic  Analysis,  p.  232. 


CHAPTER   XIV. 

EPITHELIAL  AND  CONNECTIVE  TISSUES. 
EPITHELIAL    TISSUE     (KERATIN). 

The  albuminoid  keratin  constitutes  the  major  portion  of  hair, 
horn,  hoof,  feathers,  nails  and  the  epidermal  layer  of  the  skin. 
There  is  a  g'roup  of  keratins  the  members  of  which  possess  very  sim- 
ilar properties.  The  keratins  as  a  group  are  insoluble  in  the  usual 
protein  solvents  and  are  not  acted  upon  by  the  gastric  or  pancreatic 
juices.  They  all  respond  to  the  xanthoproteic  and  Millon  reactions 
and  are  characterized  by  containing-  large  amounts  of  sulphur. 
Keratin  from  any  of  its  sources  may  be  prepared  in  a  pure  form 
by  treatment,  in  sequence,  with  artificial  gastric  juice,  artificial 
pancreatic  juice,  boiling  alcohol  and  boiling  ether,  from  twenty- 
four  to  forty-eight  hours  being  devoted  to  each  process. 

Experiments   on   Epithelial   Tissue. 

Keratin. 

Horn  shavings  or  nail  parings  may  be  used  in  the  experiments 
which  follow : 

i.  Solubility. — Test  the  solubility  of  keratin  in  the  ordinary  sol- 
vents (see  p.  23). 

2.  Millon' s  Reaction. 

3.  Xanthoproteic  Reaction. 

4.  Adamkiewicz'  s  Reaction. 

5.  Hopkins-Cole  Reaction. 

6.  Test  for  Loosely  Combined  Sulphur. 


CONNECTIVE   TISSUE. 

I.     WHITE   FIBROUS   TISSUE. 

The  principal  solid  constituent  of  white  fibrous  connective  tissue 
is  the  albuminoid  collagen.  This  body  is  also  found  in  smaller 
percentage  in  cartilage,  bone  and  ligament,  but  the  collagen  from 
the  various  sources  is  not  identical  in  composition.     In  common  with 


228  PHYSIOLOGICAL    CHEMISTRY. 

the  keratins,  collagen  is  insoluble  in  the  usual  protein  solvents.  It 
differs  from  keratin  in  containing  less  sulphur.  One  of  the  chief 
characteristics  of  collagen  is,  according  to  Hofmeister,  the  property 
of  being  hydrolyzed  by  boiling  acid  or  water  with  the  formation 
of  gelatin.  Emmett  and  Gies  claim  that  under  these  conditions 
there  is  an  intramolecular  rearrangement  of  collagen  and  the  re- 
sultant gelatin  is  consequently  not  the  product  of  hydrolysis.  The 
liberation  of  ammonia  from  the  collagen  during  the  process  ap- 
parently confirms  this  view.  Collagen  gives  Millon's  reaction  as 
well  as  the  xanthoproteic  and  biuret  tests. 

The  form  of  white  fibrous  tissue  most  satisfactory  for  general 
experiments  is  the  tendo  Achillis  of  the  ox.  According  to  Buerger 
and  Gies  the  fresh  tissue  has  the  following  composition : 

Water     62.87  % 

Solids    37.13 

Inorganic  matter 0.47 

Organic  matter 36.66 

Fatty  substance  (ether-soluble) 1.04 

Coagulable  protein 0.22 

Mucoid  1.28 

Elastin    1.63 

Collagen    31-59 

Extractives,  etc 0.90 

The  mucoid  mentioned  above  is  called  tendomucoid  and  is  a  gly- 
coprotein. It  possesses  properties  similar  to  those  of  other  con- 
nective tissue  mucoids,  c.  g.,  osseomucoid  and  chondromucoid. 

Gelatin,  the  body  which  results  from  the  hydrolysis  of  collagen 
(see  statement  of  Emmett  and  Gies  above)  is  also  an  albuminoid. 
It  responds  to  nearly  all  the  protein  tests.  It  differs  from  the 
keratins  and  collagen  in  being  easily  digested  and  absorbed.  Gel- 
atin is  not  a  satisfactory  substitute  for  the  protein  constituents  of 
a  normal  diet  however,  since  a  certain  portion  of  its  nitrogen  is 
not  available  for  the  uses  of  the  organism.  Gelatin  from  cartilage 
differs  from  the  gelatin  from  other  sources  in  containing  a  lower 
percentage  of  nitrogen.  Tyrosine  and  tryptophane  are  not  num- 
bered among  the  decomposition  products  of  gelatin,  hence  it  does 
not   respond  to   Millon's   reaction   or  the   Hopkins-Cole   reaction. 

Experiments  on  White  Fibrous  Tissue. 

The  tendo  Achillis  of  the  ox  may  be  taken  as  a  satisfactory  type 
of  the  white  fibrous  connective  tissue. 

1.  Preparation  of  Tendomucoid. — Dissect  away  the  fascia  from 


EPITHELIAL    AND    CONNECTIVE    TISSUES.  229 

about  the  tendon  and  cut  the  clean  tendon  into  small  pieces.  Wash 
the  pieces  in  water,  changing  the  wash  water  often  in  order  to 
remove  as  much  as  possible  of  the  soluble  protein  and  inorganic 
salts.  Transfer  the  washed  pieces  of  tendon  to  a  flask  and  add 
300  c.c.  of  half-saturated  lime  water.1  Shake  the  flask  at  intervals 
for  twenty-four  hours.  Filter  off  the  pieces  of  tendon  and  pre- 
cipitate the  mucoid  with  dilute  hydrochloric  acid.  Allow  the  mu- 
coid precipitate  to  settle,  decant  the  supernatant  fluid  and  filter  the 
remainder.     Test  the  mucoid  as  follows. 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see 
page  23). 

(b)  Biuret  Test. — First  dissolve  the  mucoid  in  potassium  hydrox- 
ide solution  and  then  add  a  dilute  solution  of  cupric  sulphate. 

(c)  Test  for  Loosely  Combined  Sulphur. 

(d)  Hydrolysis  oi  Tendomucoid. — Place  the  remainder  of  the 
mucoid  in  a  small  beaker,  add  about  30  c.c.  of  water  and  2  c.c. 
of  dilute  hydrochloric  acid  and  boil  until  the  solution  becomes  dark 
brown.  Cool  the  solution,  neutralize  it  with  solid  potassium  hy- 
droxide and  test  by  Fehling's  test.  With  a  reduction  of  Fehling's 
solution  and  a  positive  biuret  test  what  do  you  conclude  regarding 
the  nature  of  tendomucoid? 

2.  Collagen. — This  substance  is  present  in  the  tendon  to  the  ex- 
tent of  about  32  per  cent.  Therefore  in  making  the  following 
tests  upon  the  pieces  of  tendon  from  which  the  mucoid,  soluble 
protein  and  inorganic  salts  were  removed  in  the  last  experiment, 
we  may  consider  the  tests  as  being  made  upon  collagen. 

(a)  Solubility. — Cut  the  collagen  into  very  fine  pieces  and  try 
its  solubility  in  the  ordinary  solvents  (see  page  23). 

(b)  Milton's  Reaction. 

(c)  Biuret  Test. 

(d)  Xanthoproteic  Reaction. 

(e)  Hopkins-Cole  Reaction. 

(/)  Test  for  Loosely  Combined  Sulphur. — Take  a  large  piece 
of  collagen  in  a  test-tube  and  add  about  5  c.c.  of  potassium  hy- 
droxide solution.  Heat  until  the  collagen  is  partlv  decomposed, 
then  add  1-2  drops  of  plumbic  acetate  and  again  heat  to  boiling. 

(g)  Formation  of  Gelatin  from  Collagen. — Transfer  the  remain- 
der of  the  pieces  of  collagen  to  a  casserole,  fill  the  vessel  about  two- 
thirds  full  of  water  and  boil  for  several  hours,  adding  water  at 

'Made  by  mixing  equal  volumes  of  saturated  lime-water  and  water  from  the 
faucet. 


23O  PHYSIOLOGICAL    CHEMISTRY. 

intervals  as  needed.     By  this  means  the  collagen  is  transformed  and 
a  body  known  as  gelatin  is  produced  (see  p.  228). 

3.  Gelatin. — On  the  gelatin  formed  from  the  transformation  of 
collagen  in  the  above  experiment  (g),  or  on  gelatin  furnished  by  the 
instructor  make  the  following  tests : 

(a)  Solubility. — Try  the  solubility  in  the  ordinary  solvents  (see 
page  23)  and  in  hot  water. 

(b)  Millon's  Reaction. 

(c)  Hopkins-Cole  Reaction. — Conduct  this  test  according  to 
the  modification  given  on  page  101. 

(d)  Test  for  Loosely  Combined  Sulphur. 

Make  the  following  tests  upon  a  solution  of  gelatin  in  hot  water : 

(a)  Precipitation  by  Mineral  Acids. — Is  it  precipitated  by  strong 
mineral  acids  such  as  concentrated  hydrochloric  acid? 

(b)  Salting-out  Experiment. — Saturate  a  little  of  the  solution 
with  solid  ammonium  sulphate.  Is  the  gelatin  precipitated?  Re- 
peat the  experiment  with  sodium  chloride.     What  is  the  result? 

(c)  Precipitation  by  Metallic  Salts. — Is  it  precipitated  by  metal- 
lic salts  such  as  cupric  sulphate,  mercuric  chloride  and  plumbic 
acetate  ? 

(d)  Coagulation  Test. — Does  it  coagulate  upon  boiling? 

(e)  Precipitation  by  Alkaloidal  Reagents. — Is  it  precipitated 
by  such  reagents  as  picric  acid,  tannic  acid  and  trichloracetic  acid? 

(/)   Biuret  Test. — Does  it  respond  to  the  biuret  test? 

(g)  Bardach's  Reaction. — Does  it  yield  the  typical  crystals  of 
this  reaction?     (See  page  94.) 

(h)  Precipitation  by  Alcohol. — Fill  a  test-tube  one-half  full  of 
95  per  cent  alcohol  and  pour  in  a  small  amount  of  concentrated 
gelatin  solution.  Do  you  get  a  precipitate?  How  would  you  pre- 
pare pure  gelatin  from  the  tendo  Achillis  of  the  ox? 

II.     YELLOW    ELASTIC    TISSUE     (ELASTIN). 

The  Ligamentum  nuchce  of  the  ox  may  be  taken  as  a  satisfactory 
type  of  the  yellow  elastic  connective  tissue.  The  principal  solid 
constituent  of  this  tissue  is  clastin,  a  member  of  the  albuminoid 
group.  In  common  with  the  keratins  and  collagen,  elastm  is  an 
insoluble  body  and  gives  the  protein  color  reactions.  It  differs 
from  keratin  principally  in  the  fact  that  it  may  be  digested  by 
enzymes  and  that  it  contains  a  very  small  amount  of  sulphur. 


EPITHELIAL   AND    CONNECTIVE    TISSUES.  23  I 

Yellow  elastic  tissue  also  contains  mucoid  and  collagen  but  these 
are  present  in  much  smaller  amount  than  in  white  fibrous  tissue, 
as  may  be  seen  from  the  following  percentage  composition  of 
the  fresh  ligamentum  nuchas  of  the  ox  as  determined  by  Vandegrift 
and  Gies : 

Water     57-57% 

Solids    42.43 

Inorganic  matter   0.47 

Organic  matter  41.96 

Fatty  substance    (ether-soluble) 1.12 

Coagulable  protein 0.62 

Mucoid     0.53 

Elastin    31.67 

Collagen     7.23 

Extractives,  etc 0.80 

Experiments    on     Elastin. 

1.  Preparation  of  Elastin  (Richards  and  Gies). — Cut  the  lig- 
ament into  fine  strips,  run  it  through  a  meat  chopper  and  wash 
the  finely  divided  material  in  cold,  running  wTater  for  24-48  hours. 
Add  an  excess  of  half -saturated  lime  water  (see  note  at  bottom 
of  p.  229)  and  allow  the  hashed  ligament  to  extract  for  48-72 
hours.  Decant  the  lime-water,  remove  all  traces  of  alkali  by  wash- 
ing in  water  and  then  boil  in  water  with  repeated  renewals  until 
only  traces  of  protein  material  can  be  detected  in  the  wrash  water. 
Decant  the  fluid  and  boil  the  ligament  in  10  per  cent  acetic  acid 
for  a  few  hours.  Treat  the  pieces  with  5  per  cent  hydrochloric 
acid  at  room  temperature  for  a  similar  period,  extract  again  in  hot 
acetic  acid  and  in  cold  hydrochloric  acid.  Wash  out  traces  of  acid 
by  means  of  water  and  then  thoroughly  dehydrolyze  by  boiling  al- 
cohol and  boiling  ether  in  turn.  Dry  in  an  air-bath  and  grind  to 
a  powder \  in  a  mortar. 

2.  Solubility. — Try  the  solubility  of  the  finely  divided  elastin, 
prepared  by  yourself  or  furnished  by  the  instructor,  in  the  ordinary 
solvents  (see  page  23).  How  does  its  solubility  compare  with 
that  of  collagen? 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Biuret  Test. 

6.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  101. 

7.  Test  for  Loosely  Combined  Sulphur. 


232  PHYSIOLOGICAL    CHEMISTRY. 

III.     CARTILAGE. 

The  principal  solid  constituents  of  the  matrix  of  cartilaginous 
tissue  are  chrondromucoid,  chrondroitin-sulphuric  acid,  chrondroal- 
bumoid  and  collagen.  Chrondromucoid  differs  from  the  mucoids 
isolated  from  other  connective  tissues  in  the  large  amount  of  chron- 
droitin-sulphuric acid  obtained  upon  decomposition.  Besides  being 
an  important  constituent  of  all  forms  of  cartilage,  chrondroitin- 
sulphuric  acid  has  been  found  in  bone,  ligament,  the  mucosa  of  the 
pig's  stomach,  the  kidney  of  the  ox,  the  inner  coats  of  large  arteries 
and  in  human  urine.  It  may  be  decomposed  through  the  action  of 
acid  and  yields  a  nitrogenous  body  known  as  chrondroitin  and  later 
this  body  yields  chrondrosin.  Chrondrosin  is  also  a  nitrogenous 
body  and  has  the  power  of  reducing  Fehling's  solution  more  strongly 
than  dextrose.  Sulphuric  acid  is  a  by-product  in  the  formation  of 
chrondroitin,  and  acetic  acid  is  a  by-product  in  the  formation  of 
chrondrosin. 

Chrondroalbumoid  is  similar  in  some  respects  to  elastin  and 
keratin.  It  differs  from  keratin  in  being  soluble  in  gastric  juice  and 
in  containing  considerably  less  sulphur  than  any  member  of  the 
keratin  group.     It  gives  the  usual  protein  color  reactions. 

Experiments    on    Cartilage. 

1.  Preparation  of  the  Cartilage. — Boil  the  trachea  of  an  ox  in 
water  until  the  cartilage  rings  may  be  completely  freed  from  the 
surrounding  tissue.  Use  the  cartilage  so  obtained  in  the  following 
experiments. 

2.  Solubility. — Cut  one  of  the  rings  into  very  small  pieces  and 
try  the  solubility  of  the  cartilage  in  the  ordinary  solvents  (see  page 

23)- 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Hopkins-Cole  Reaction. — Conduct  this  test  according  to  the 
modification  given  on  page  101. 

6.  Test  for  Loosely  Combined  Sulphur. 

7.  Preparation  of  Cartilage  Gelatin. — Cut  the  remaining  carti- 
lage rings  into  small  pieces,  place  them  in  a  casserole  with  water 
and  boil  for  several  hours.  Filter  while  the  solution  is  still  hot. 
Observe  that  the  filtrate  soon  becomes  more  or  less  solid.  What 
is  the  reason  for  this  ?  Bring  a  portion  of  the  material  into  solution 
by  heat  and  try  the  following  tests : 


EPITHELIAL    AND    CONNECTIVE    TISSUES.  233 

(a)  Biuret  Test. 

(b)  Bardach's  Reaction. 

(c)  Test  for  Loosely  Combined  Sulphur. 

(d)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few- 
drops  of  barium  chloride.  Do  you  get  a  precipitate,  and  if  so  to 
what  is  the  precipitate  due  ? 

(e)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few 
drops  of  dilute  hydrochloric  acid  and  boil  for  a  few  moments. 
Now  add  a  little  barium  chloride  to  this  solution.  Is  the  precipi- 
tate any  larger  than  that  obtained  in  the  preceding  experiment? 
Why? 

(/)  To  the  remainder  of  the  solution  add  a  little  dilute  hydro- 
chloric acid  and  boil  for  a  few  moments.  Cool  the  solution,  neu- 
tralize with  solid  potassium  hydroxide  and  try  Fehling's  test.  Ex- 
plain the  result. 

IV.     OSSEOUS    TISSUE. 

Bone  is  composed  of  about  equal  parts  of  organic  and  inorganic 
matter.  The  organic  portion,  called  ossein,  may  be  obtained  by 
removing  the  inorganic  salts  through  the  medium  of  dilute  acid. 
Ossein  is  practically  the  same  body  which  is  termed  collagen  in 
the  other  connective  tissues,  and  in  common  with  collagen  yields 
gelatin  upon  being  boiled  with  dilute  mineral  acid. 

In  common  with  the  other  connective  tissues  bone  contains  a 
mucoid  and  an  albumoid.  Because  of  their  origin  these  bodies 
are  called  osseomucoid  and  osseoalbumoid.  Osseomucoid,  when 
boiled  with  hydrochloric  acid,  yields  sulphuric  acid  and  a  sub- 
stance capable  of  reducing  Fehling's  solution.  The  composition 
of  osseomucoid  is  very  similar  to  that  of  tendomucoid  and  chron- 
dromucoid  (see  page  106). 

Experiment   on    Osseous    Tissue. 

Analysis  of  Bone  Ash. — Take  one  gram  of  bone  ash  in  a  small 
beaker  and  add  a  little  dilute  nitric  acid.  What  does  the  effer- 
vescence indicate?  Stir  thoroughly  and  when  the  major  portion 
of  the  ash  is  dissolved  add  an  equal  volume  of  water  and  filter. 
To  the  acid  nitrate  add  ammonium  hydroxide  to  alkaline  reaction. 
A  heavy  white  precipitate  of  phosphates  results.  (What  phos- 
phates are  precipitated  here  by  the  ammonia?)  Filter  and  test  the 
filtrate  for  chlorides,  sulphates,  phosphates  and  calcium.  Add 
dilute  acetic  acid  to  the  precipitate  on  the  paper  and  test  this  filtrate 


234 


PHYSIOLOGICAL    CHEMISTRY. 


for  calcium  and  phosphates.  To  the  precipitate  remaining  un- 
dissolved on  the  paper  add  a  little  dilute  hydrochloric  acid  and  test 
this  last  filtrate  for  phosphates  and  iron. 

Reference  to  the  following  scheme  may  facilitate  the  analysis. 

BONE    ASH. 

I 

Add  dilute  nitric  acid,  stir  thoroughly  and  after  the  major  portion  of  the  ash 
has  been  brought  into  solution  add  a  little  distilled  water  and  filter. 


Residue  I.  Filtrate  I. 

(discard)  Add   ammonium  hy- 

droxide to  alkaline  re- 
action and  filter. 


Residue  II. 

Treat  on  paper  with  acetic  acid. 


Residue  III. 
Treat  on  paper  with 
hydrochloric  acid. 

I 

Filtrate  IV. 
Test  for : 
i.  Iron. 
2.  Phosphates. 


Filtrate  HI. 
Test  for: 

i.  Phosphates. 
2.  Calcium. 


Filtrate  II. 

Test  for : 

1.  Chlorides. 

2.  Sulphates. 

3.  Phosphates. 

4.  Calcium. 


V.     ADIPOSE  TISSUE. 
For  discussion  and  experiments  see  the  chapter  on  Fats,  page  131. 


CHAPTER   XV. 

MUSCULAR  TISSUE. 

The  muscular  tissues  are  divided  physiologically  into  the  vol- 
untary (striated)  and  the  involuntary  (nonstriated).  In  the  chem- 
ical examination  of  muscular  tissue  the  voluntary  form  is  generally 
employed.  Muscle  contains  about  25  per  cent  of  solid  matter, 
of  which  about  four-fifths  is  protein  material  and  the  remaining 
one-fifth  extractives  and  inorganic  salts. 

The  proteins  are  the  most  important  of  the  constituents  of  mus- 
cular tissue.  In  the  living  muscle  we  find  two  proteins,  myosino- 
gen and  para-myosinogen.  These  may  be  shown  to  be  present  in 
muscle  plasma  expressed  from  fresh  muscles.  In  common  with  the 
plasma  of  the  blood  this  muscle  plasma  has  the  power  of  coagulat- 
ing, and  the  clot  formed  in  this  process  is  called  myosin.  In  the 
onset  of  rigor  mortis  we  have  an  indication  of  the  formation  of  this 
myosin  clot  within  the  body.  The  relation  between  the  proteins 
of  living  and  dead  muscle  is  represented  graphically  by  Halliburton 
as   follows : 

Proteins   of  the   living  muscle. 


Para-myosinogen    (25%).  Myosinogen  (75%). 

Soluble  myosin. 


Myosin. 
(The  protein  of  the  muscle  clot.) 

Of  the  total  protein  content  of  living  muscle  about  75  per  cent 
is  made  up  by  the  myosinogen  and  the  remaining  25  per  cent  is 
para-myosinogen.  These  proteins  may  be  separated  by  subjecting 
the  muscle  plasma  to  fractional  coagulation  in  the  usual  way. 
Under  these  conditions  the  para-myosinogen  is  found  to  coagulate 
at  470  C.  and  the  myosinogen  to  coagulate  at  560  C.  It  is  also 
claimed  by  some  investigators  that  it  is  possible  to  separate  these 
two  proteins  by  the  fractional  ammonium  sulphate  method,  but 
the  possibility  of  making  an  accurate  separation  by  this   method 

235 


236  PHYSIOLOGICAL    CHEMISTRY. 

is  somewhat  doubtful.  It  is  well  established  that  para-myosino- 
gen  is  a  globulin  since  it  responds  to  certain  of  the  protein  precipi- 
tation tests  and  is  insoluble  in  water.  Myosinogen,  on  the  con- 
trary, is  not  a  typical  globulin  since  it  is  soluble  in  water.  It  has 
been  called  a  pseudo-globulin.  Myosin  possesses  the  globulin  char- 
acteristics. It  is  insoluble  in  water  but  soluble  in  the  other  pro- 
tein solvents  and  is  precipitated  from  its  solution  upon  saturation 
with  sodium  chloride. 

Very  recently  Mellanby  has  reported  observations  which  he 
claims  indicate  that  there  is  only  one  protein  in  muscle  and  that 
rigor  mortis  is  due  to  the  coagulation  of  this  protein  under  the 
combined  influences  of  the  salt  present  in  the  muscle  and  the  lac- 
tic acid  developed  upon  the  death  of  the  muscle.  He  further  states 
that  the  disappearance  of  rigor  is  due  to  the  fact  that  the  lactic  acid 
which  is  continually  formed  brings  this  protein  into  solution. 

Under  the  name  extractives  we  class  a  number  of  muscle  con- 
stituents which  occur  in  traces  in  the  tissue  and  may  be  extracted 
by  water,  alcohol  or  ether.  There  are  two  classes  of  these  extrac- 
tives, the  non-nitrogenous  extractives  and  the  nitrogenous  extrac- 
tives. Grouped  under  the  non-nitrogenous  bodies  we  have  glyco- 
gen, dextrin,  sugars,  lactic  acid,  inosite,  C6H6(OH)6,  and  fat.  In 
the  class  of  nitrogenous  extractives  we  have  creatine,  creatinine, 
xanthine,  hypoxanthine,  uric  acid,  urea,  carnine,  guanine,  phospho- 
carnic  acid,  inosinic  acid,  carnosine,  taurine,  carnitine,  novaine,  ig- 
notine,  neosine,  oblitine,  carnomuscarine  and  methyl guanidine  (see 
formulas  on  page  240).  Not  all  of  these  extractives  are  present  in 
the  muscles  of  all  species  of  animals.  Other  extractives  besides 
those  enumerated  above  have  been  described  and  there  are  undoubt- 
edly still  others  whose  presence  remains  undetermined.  A  detailed 
consideration  would  however  be  unprofitable  in  this  place. 

Glycogen  is  an  important  constituent  of  muscle.  The  content 
of  this  polysaccharide  in  muscle  varies  and  is  markedly  decreased 
by  intense  muscular  activity.  It  is  transformed  into  sugar 
and  used  as  fuel.  The  liver  is  the  organ  which  stores  the  re- 
serve supply  of  glycogen  and  transforms  it  into  dextrose  which  is 
passed  into  the  blood  stream  and  so  carried  to  the  working  muscle 
where  it  is  synthesized  into  glycogen.  The  glycogen  thus  formed  is 
then  changed  into  dextrose  as  the  working  muscle  may  need  it. 

Glycogen  is  a  polysaccharide  and  has  the  same  percentage  com- 
position as  starch  and  dextrin.  It  resembles  starch  in  forming  an 
opalescent  solution  and  resembles  dextrin  in  being  very  soluble, 


MUSCULAR    TISSUE.  237 

in  giving  a  reddish  color  with  iodine  and  in  being  dextro-rotatory. 
Glycogen  may  be  prepared  from  muscle  by  extracting  with  boiling 
water  and  then  precipitating  the  glycogen  from  the  aqueous  solu- 
tion by  alcohol :  dilute  or  concentrated  potassium  hydroxide  may 
also  be  used  to  extract  the  glycogen.  Glycogen  may  be  prepared 
in  the  form  of  a  white,  tasteless,  amorphous  powder.  It  is  com- 
pletely precipitated  from  its  solution  by  saturation  with  solid  am- 
monium sulphate,  but  is  not  precipitated  by  saturation  with  sodium 
chloride.  It  may  also  be  precipitated  by  alcohol,  tannic  acid  or 
ammoniacal  basic  lead  acetate.  It  has  the  power  of  holding  cupric 
hydroxide  in  solution  in  alkaline  fluids  but  cannot  reduce  it.  It 
may  be  hydrolyzed  with  the  formation  of  dextrose  by  dilute  min- 
eral acids  and  is  readily  digested  by  amylolytic  enzymes. 

Mendel  and  Leavenworth  have  recently  drawn  the  conclusion, 
from  the  examination  of  embryo  pigs,  that  embryonic  structures 
do  not  contain  exceptionally  large  amounts  of  glycogen.  The  dis- 
tribution of  the  glycogen  was  not  observed  to  differ  from  that  in 
the  adult  animal  except  that  the  liver  of  the  embryo  does  not 
assume  its  glycogen-storing  function  early.  They  further  draw 
the  conclusion  that  the  metabolic  transformations  of  glycogen  in 
the  embryo  and  the  adult  are  entirely  analogous. 

The  lactic  acid  occurring  in  the  muscular  tissue  of  vertebrates 
is  paralactic  or  sarcolactic  acid, 

H    OH 

H-C-C-COOH. 

H   H 

The  reaction  of  an  inactive  living  muscle  is  alkaline,  but  upon  the 
death  of  the  muscle,  or  after  the  continued  activity  of  a  living 
muscle,  the  reaction  becomes  acid,  due  to  the  formation  of  lactic 
acid.  There  is  a  difference  of  opinion  regarding  the  origin  of  this 
lactic  acid.  Some  investigators  claim  it  to  arise  from  the  carbo- 
hydrates of  the  muscle,  while  others  ascribe  to  it  a  protein  origin. 

Among  the  nitrogenous  extractives  of  muscle,  those  which  are 
of  the  most  interest  in  this  connection  are  creatine  and  the  purine 
bases,  xanthine  and  hypoxanthine.  Creatine  is  found  in  varying 
amounts  in  the  muscles  of  different  species,  the  muscles  of  birds 
having  shown  the  largest  amount.  It  has  also  been  found  in  the 
blood,  the  brain,  in  transudates  and  in  the  thyroid  gland.     Creatine 


238 


PHYSIOLOGICAL    CHEMISTRY. 


may  be  crystallized  and  forms  colorless  rhombic  prisms   (Fig.  JJ, 
below)  which  are  soluble  in  warm  water  and  practically  insoluble 


Fig.    77. 


Creatine. 


in  alcohol  and  ether.  Upon  boiling  a  solution  of  creatine  with 
dilute  hydrochloric  acid  it  is  dehydrolyzed  and  its  anhydride  crea- 
tinine is  formed.  The  theory  that  the  creatine  of  ingested  meat  is 
transformed  into  creatinine  and  excreted  in  the  urine  has  been 
proven  untenable  through  the  recent  researches  of  Folin,  Klercker, 
and  Wolf  and  Shaffer.  It  is  now  known  that  the  ingestion  of 
creatine  in  no  way  influences  the  excretion. of  creatinine.  In  this 
connection  it  is  important  to  note  that  there  is  no  normal  excretion 
of  endogenous  (see  p.  272)  creatine,  a  statement  proven  by  the 
fact  that  if  no  creatine  be  ingested  none  will  be  excreted.  Folin 
has  shown  that  the  main  bulk  of  ingested  creatine  is  retained  in  the 
body,  unless  the  diet  contains  a  large  amount  of  protein  material. 
Under  pathological  conditions  the  urine  contains  endogenous  crea- 
tine which  is  probably  derived  from  the  catabolism  of  muscular 
tissue,  as  Benedict,  Mellanby,  and  Shaffer  have  suggested. 

Besides  being  a  normal  constituent  of  muscle,  xanthine  has  been 
found  in  the  brain,  spleen,  pancreas,  thymus,  kidneys,  testicles, 
liver,  and  in  the  urine.  It  may  be  obtained  in  crystalline  form 
(Fig.  78,  p.  239)  but  ordinarily  it  is  amorphous.  Xanthine  is  easily 
soluble  in  alkalis,  less  easily  soluble  in  water  and  dilute  acids,  and 
entirely  insoluble  in  alcohol  and  ether. 

Hypoxanthine  occurs  ordinarily  in  those  tissues  and  fluids  which 


MUSCULAR    TISSUE. 


239 


contain  xanthine.  It  lias  been  found  unaccompanied  by  xanthine, 
in  bone  marrow  and  in  milk.  Unlike  xanthine  it  may  be  easily 
crystallized  in  the  form  of  small,  colorless  needles.  It  is  readily 
soluble  in  alkalis,  acids  and  boiling  water,  less  soluble  in  cold  water 
and  practically  insoluble  in  alcohol  and  ether. 

The  predominating  inorganic  salt  of  muscle  is  potassium  phos- 
phate. Besides  this  salt  we  have  present  chlorides  and  salts  of 
sodium,  calcium,  magnesium  and  iron.  Sulphates  are  also  present 
in  traces. 

Mendel  and  Saiki  have  recently  made  some  interesting  observa- 
tions upon  the  chemical  composition  of  nominated  (involuntary) 
mammalian  muscle,  such  as  the  urinary  bladder  and  the  muscular 
coat  of  the  stomach  of  the  pig.  Hypoxanthine  was  found  to  be 
the  predominant  purine  base  present.  Creatine  and  paralactic  acid 
were  also  isolated.  These  investigators  were  unable  to  demonstrate, 
definitely,  the  presence  of  glycogen  in  the  nonstriated  muscles 
studied,  but  state  that  "  the  tissues  possess  the  property  of  trans- 

Fig.    78. 


Xanthine. 

After  tne  drawings  of  Horbaczewski,  as  represented  in  Neubauer  and  Yogel. 

(Ogden.) 


forming  glycogen  in  the  characteristic  enzymatic  way."  The  most 
important  part  of  their  investigation  consists  in  a  rather  complete 
analysis  of  the  inorganic  constituents  of  these  muscles.  A  notable 
difference  in  the  relative  distribution  of  the  various  inorganic  con- 
stituents was  observed,  a  difference  which,  according  to  the  authors, 
"can  be  accounted  for  in  part  only  by  an  admixture  of  lymph." 
The  comparative  composition  of  the  inorganic  portion  of  striated 


240 


PHYSIOLOGICAL    CHEMISTRY. 


and  nonstriated  muscle  and  of  blood  serum  for  comparison  is  shown 
in  the  appended  table : 


K20 

Na20 

Fe203 

CaO 

MgO 

CI 

p2o6 

H20 

Nonstriated  muscle   (Mendel 

O.081 
0.306 
O.027 

O.328 
0.2IO 
O.425 

O.OII 

O.O08 

O.O44 
O.OII 
O.OI2 

O.O07 
O.047 
O.OO4 

0. 171 

O.048 
O.363 

0.184 

0.487 

0.020 

80.6 

Blood  serum  (Abderhalden)f.. 

72.9 
91.8 

Muscular  tissue  is  said  to  contain  a  reddish  pigment  called  myo- 
hcematin,  which  is  a  derivative  of  haemoglobin. 

The  so-called  "  fatigue  substances  "  of  muscle  are  carbon  diox- 
ide, paralactic  acid  and  potassium  dihydrogen  phosphate. 

The  ordinary  commercial  "meat  extract"  is  composed  princi- 
pally of  the  water-soluble  constituents  of  muscle  and  contains  practi- 
cally nothing  of  nutritive  value.  The  protein  material  to  which 
meat  owes  its  value  as  an  article  of  diet  is  practically  all  removed 
in  the  preparation  of  the  extract. 

The  structural  formulas  of  the  nitrogenous  extractives  of  muscle 
are  as  follows : 


NIL 


HN  =  C 

NCH3CH2COOH 

Creatine,  C4H9N3O2. 

M ethyl- guanid 'in e  acetic  acid. 


HN- 


-C0 


HN-C 


N-CH8-CHS 

Creatinine,  C4H7N3O. 
Creatine  anhydride. 


NH, 


0 


NH2 

Urea,  CON2H4. 


CH2NH2 

I 
CH2S02OH 

Taurine,  C2H7NS03. 
Amino-ethyl-sulphonic  acid. 


0- 


(CH3)3-N 


/ 

r 
\ 


-co 


CH2-CHOH-CH2 

Carnitine,  C7H15NO3. 
'y-trimethyloxybutyrobetaine. 


Carnosine,  C9H14N40< 


MUSCULAR    TISSUE. 


24I 


Neosine,  C6H17N02. 

Novaine,  C7H17N02. 

Ignotine,  CgH^N^O;,. 

Phosphocarnic  acid,  C10H17N3O5  or  C10H15N3O5. 

Inosinic  acid,  (HO)2POOCH2(CHOH)3CH:  (CbH3N40). 

The  following-  extractives  as  a  group  are  called  purine  bodies. 
Their  formulas,  together  with  that  of  purine  from  which  they  are 
derived  and  the  hypothetical  "  purine  nucleus  "  follow  : 


N=CH 
HC    C-NH 


N-C-N 

Purine,  C5H4N4. 

HN-CO 
HC    C-NH 


>CH 


N-C-N 


/ 


CH 


Hypoxanthine,  C5H4N40. 

6-oxypurine. 

HN-CO 

OC     C-NH 
I       II        \ 


I       II         / 
HN-C-NH 

Uric  Acid,  C5H4N4O3. 
2-6-8-trioxyp  u  rin  e. 


CO 


'N-C6 

,V     C5-N- 


\c, 


3N-G4-N, 

Purine  Nucleus. 

HN-CO 
OC     C-NH 


/ 


CH 


HN-C-N 

Xanthine,  CsH^XiOn 
2-6-dioxypurin  e. 

N-CNHo 

HC     C-NH 
\ 


/ 


CH 


N-C-N 

Adenine,  C5H5Nt3. 
6-aminopurine. 


HN-CO 

H„NC     C-NH 

II      II        \ 


N-C-N 

Guanine,  C5H.-,X.-,0. 
2-amino-6-oxy  purine. 

Experiments  on  Muscular  Tissue. 

I.    Experiments  on  "  Living  "  Muscle. 

i.  Preparation  of  Muscle  Plasma  (Halliburton). — Wash  out 
the  blood  vessels  of  a  freshly  killed  rabbit  with  0.9  per  cent  sodium 
17 


242  PHYSIOLOGICAL    CHEMISTRY. 

chloride.  This  can  best  be  done  by  opening  the  abdomen  and  in- 
serting a  cannula  into  the  aorta.  Now  remove  the  skin  from  the 
lower  limbs,  cut  away  the  muscles  and  divide  them  into  very  small 
pieces  by  means  of  a  meat  chopper.  Transfer  the  pieces  of  muscle 
to  a  mortar  and  grind  them  with  clean  sand  and  a  little  5  per  cent 
magnesium  sulphate.  Filter  off  the  salted  muscle  plasma  and  make 
the  following  tests : 

(a)  Reaction. — Test  the  reaction  to  litmus.  What  is  the  reac- 
tion of  this  fresh  muscle  plasma? 

(b)  Fractional  Coagulation. — Place  a  little  muscle  plasma  in  a 
test-tube  and  arrange  the  apparatus  for  fractional  coagulation  as 
explained  on  page  100.  Raise  the  temperature  very  carefully  from 
30 °  C.  and  note  any  changes  which  may  occur  and  the  exact  tem- 
perature at  which  such  changes  take  place.  When  the  first  protein 
(para-myosinogen)  coagulates  filter  it  off  and  then  heat  the  clear 
filtrate  as  before,  being  careful  to  note  the  exact  temperature  at 
which  the  next  coagulation  (myosinogen)  occurs.  There  will 
probably  be  a  preliminary  opalescence  in  each  case  before  the  real 
coagulation  occurs.  Therefore  do  not  mistake  the  real  coagulation- 
point  and  filter  at  the  wrong  time.  What  are  the  coagulation  tem- 
peratures of  these  two  proteins?  Which  protein  was  present  in 
greater  amount? 

(c)  Formation  of  the  Myosin  Clot. — Dilute  a  portion  of  the 
plasma  with  3  or  4  times  its  volume  of  water  and  place  it  on  a 
water-bath  or  in  an  incubator  at  35 °  C.  for  several  hours.  A 
typical  myosin  clot  should  form.  Note  the  muscle  serum  surround- 
ing the  clot.  Now  test  the  reaction.  Has  the  reaction  changed, 
and  if  so  to  what  is  the  change  due?  Make  a  test  for  lactic  acid. 
What  do  you  conclude? 

2.  Preparation  of  Muscle  Plasma  (v.  Fiirth). — Remove  the 
blood-free  muscles  of  a  rabbit  as  explained  on  page  241.  Finely 
divide  by  means  of  a  meat  chopper  and  grind  in  a  mortar  with  a 
little  clean  sand  and  some  0.9  per  cent  sodium  chloride.  Wrap 
portions  of  the  muscle  in  muslin  and  press  thoroughly  by  means 
of  a  tincture  press  or  lemon  squeezer.  Filter  and  make  the  tests 
according  to  the  directions  given  in  the  last  experiment. 

3.  "  Fuchsin-Frog  "  Experiment. — Inject  a  saturated  aqueous 
solution  of  Fuchsin  "  S  "  into  the  lymph  spaces  of  a  frog  three  or 
four  times  daily  for  two  or  three  days,  in  this  way  thoroughly  satu- 
rating the  tissues  with  the  dye.  Pith  the  animal  (insert  a  heavy 
wire  or  blunt  needle  through  the  occipito  atlantoid  membrane),  re- 


MUSCULAR    TISSUE.  243 

move  the  skin  from  both  hind  legs  and  expose  the  sciatic  nerve  in 
one  of  them.  Insert  a  small  wire  hook  through  the  jaws  of  the 
frog  and  suspend  the  animal  Erom  an  ordinary  clamp  or  iron  ring. 
Pass  electrodes  under  the  exposed  sciatic  nerve,  and  after  tying 
the  other  leg  to  prevent  any  muscular  movement,  stimulate  the 
exposed  nerve  by  means  of  make  and  break  shocks  from  an  induc- 
tion coil.  The  stimulated  leg  responds  by  pronounced  muscular  con- 
tractions, whereas  the  tied  leg  remains  inactive.  Continue  the  stim- 
ulation until  the  muscles  are  fatigued.  The  muscular  activity  has 
caused  the  production  of  lactic  acid  and  this  in  turn  has  reacted 
with  the  injected  fuchsin  to  cause  a  pink  or  red  color  to  develop. 
The  muscles  of  the  inactive  leg  still  remain  unchanged  in  color. 

The  normal  color  of  the  Fuchsin  "  S  "  when  injected  was  red, 
but  upon  being  absorbed  it  became  colorless  through  the  action  of 
the  alkalinity  of  the  blood.  Upon  stimulating  the  muscles,  how- 
ever, as  above  explained,  lactic  acid  was  formed  and  this  acid  re- 
acted with  the  fuchsin  and  again  produced  the  original  color  of  the 

dye. 

II.  Experiments  on  "  Dead  "  Muscle. 

i.  Preparation  of  Myosin. — Take  25  grams  of  finely  divided 
lean  beef  which  has  been  carefully  washed  to  remove  blood  and 
lymph  constituents  and  place  it  in  a  beaker  with  10  per  cent  sodium 
chloride.  Stir  occasionally  for  several  hours.  Strain  off  the  meat 
pieces  by  means  of  cheese  cloth,  filter  the  solution  and  saturate  it 
with  sodium  chloride  in  substance.  Filter  off  the  precipitate  of  my- 
osin and  make  the  tests  as  given  below.  This  filtration  will  pro- 
ceed very  slowly.  Myosin  collects  as  a  film  on  the  sides  of  the 
filter  paper  and  may  be  removed  and  tested  before  the  entire  volume 
of  fluid  has  been  filtered.  If  this  precipitate  remains  for  any  length 
of  time  on  the  paper  in  contact  with  the  air  it  will  become  trans- 
formed into  the  protean  myosan.  Test  the  myosin  precipitate  as 
follows : 

(a)  Solubility. — Try  its  solubility  in  the  ordinary  solvents.  Is 
myosin  an  albumin  or  a  globulin? 

(b)  Xanthoproteic  Reaction. — See  page  91. 

(c)  Coagulation  Test. — Suspend  a  little  of  the  myosin  in  water 
in  a  test-tube  and  heat  to  boiling  for  a  few  moments.  Now  re- 
move the  suspended  material  and  try  its  solubility  in  10  per  cent 
sodium  chloride.  What  property  does  this  experiment  show  myosin 
to  possess  ? 

Test  the  filtrate  from  the  original  myosin  precipitate  as  follows : 
(a)   Biuret  Test. — What  does  this  show  ?J 


244  PHYSIOLOGICAL    CHEMISTRY. 

(b)  Place  a  little  of  the  solution  in  a  test-tube  and  heat  to  boiling. 
At  the  boiling-point  add  a  drop  of  dilute  acetic  acid  and  filter.  Test 
this  filtrate  for  proteose  with  picric  acid.  Is  any  proteose  present  ? 
Saturate  another  portion  of  the  filtrate  with  ammonium  sulphate 
and  test  for  peptone  in  the  usual  way  (see  page  114).  Do  you 
find  any  peptone?  From  your  experiments  on  "living"  and 
"  dead "  muscle  what  are  your  ideas  regarding  the  proteins  of 
muscle  ? 

2.  Preparation  of  Glycogen. — Grind  a  few  scallops  in  a  mor- 
tar with  sand.  Transfer  to  an  evaporating  dish,  add  water  and 
boil  for  20  minutes.  At  the  boiling-point  faintly  acidify  with 
acetic  acid.  Why  is  this  acid  added  ?  Filter,  and  divide  the  filtrate 
into  two  parts.  Note  the  opalescence  of  the  solution.  Test  one 
portion  of  the  filtrate  as  follows : 

(a)  Iodine  Test. — To  5  c.c.  of  the  solution  in  a  test-tube  add 
5-10  drops  of  iodine  solution  and  2-3  drops  of  10  per  cent  sodium 
chloride.  What  do  you  observe?  Is  this  similar  to  the  iodine  test 
upon  any  other  body  with  which  we  have  had  to  deal  ? 

(&)  Reduction  Test. — Does  the  solution  reduce  Fehling's  solu- 
tion? 

(c)  Hydrolysis  of  Glycogen. — Add  10  drops  of  concentrated 
hydrochloric  acid  to  10  c.c.  of  the  solution  and  boil  for  10  minutes. 
Cool  the  solution,  neutralize  with  solid  potassium  hydroxide  and 
test  with  Fehling's  solution.  Does  it  still  fail  to  reduce  Fehling's 
solution?  If  you  find  a  reduction  how  can  you  prove  the  identity 
of  the  reducing  substance? 

(d)  Influence  of  Saliva. — Place  5  c.c.  of  the  solution  in  a  test- 
tube,  add  5  drops  of  saliva  and  place  on  the  water-bath  at  400  C. 
for  10  minutes.     Does  this  now  reduce  Fehling's  solution? 

To  the  second  part  of  the  glycogen  filtrate  add  3-4  volumes  of 
«95  per  cent  alcohol.  Allow  the  glycogen  precipitate  to  settle,  de- 
cant the  supernatant  fluid,  filter  the  remainder  and  upon  the  glyco- 
gen make  the  following  tests  : 

(a)  Solubility. — Try  its   solubility  in  the  ordinary  solvents. 

(b)  Iodine  Test. — Place  a  small  amount  of  the  glycogen  in  a  de- 
pression of  a  test-tablet  and  add  2-3  drops  of  dilute  iodine  solution 
and  a  trace  of  a  sodium  chloride  solution.  The  same  wine-red 
color  is  observed  as  in  the  iodine  test  upon  the  glycogen  solution. 


MUSCULAR    TISSUE. 


245 


Separation  of  Extractives  from  Muscle. 

1.  Creatine. — Dissolve  about  10  grams  of  a  commercial  extract 
of  meat  in  200  c.c.  of  warm  water.  Precipitate  the  inorganic 
constituents  by  neutral  lead  acetate,  being  careful  not  to  add  an  ex- 
cess of  the  reagent.  Write  the  equations  for  the  reactions  taking 
place  here.  Allow  the  precipitate  to  settle,  then  filter  and  remove 
the  excess  of  lead  in  the  zvarm  filtrate  by  hydrogen  sulphide.  Filter 
while  the  solution  is  yet  warm,  evaporate  the  clear  filtrate  to  a 
syrup  and  allow  it  to  stand  at  least  48  hours  in  a  cool  place.  Crys- 
tals of  creatine  should  form  at  this  point.  Examine  under  the 
microscope  (Fig.  yy,  page  238).  Treat  the  syrup  with  200  c.c.  of 
88  per  cent  alcohol,  stir  well  with  a  glass  rod  to  bring  all  soluble 
material  into  solution,  and  then  filter.  The  purine  bases  have  been 
dissolved  and  are  in  the  filtrate,  whereas  the  creatine  crystals  were 
insoluble  in  the  88  per  cent  alcohol  and  remain  on  the  filter  paper. 
Wash  the  crystals  with  88  per  cent  alcohol,  then  remove  them  and 
bring  them  into  solution  in  a  little  hot  water.     Decolorize  the  solu- 

Fig.    79. 


Hypoxanthine  Silver  Nitrate. 


tion  by  animal  charcoal  and  concentrate  it  to  a  small  volume. 
Allow  the  solution  to  cool  and  note  the  separation  of  colorless  crys- 
tals of  creatine.  Examine  these  crystals  under  the  microscope  and 
compare  them  with  those  reproduced  in  Fig.  yy,  page  238. 

2.  Hypoxanthine. — Evaporate  the  alcoholic  filtrate  from  the 
creatine  to  remove  the  alcohol.  Make  the  solution  ammoniacal 
and  add  ammoniacal  silver  nitrate  until  precipitation  ceases.     The 


246  PHYSIOLOGICAL    CHEMISTRY. 

precipitate  consists  principally  of  hypoxanthine  silver  and  xanthine 
silver.  Collect  these  silver  salts  on  a  filter  paper  and  wash  them 
with  water.  Place  the  precipitate  and  paper  in  an  evaporating  dish 
and  boil  for  one  minute  with  nitric  acid  having  a  specific  gravity 
of  1.1.  Filter  while  hot  through  a  double  paper,  wash  with  the 
same  strength  of  nitric  acid  and  allow  the  solution  to  cool.  By 
this  treatment  with  nitric  acid  hypoxanthine  silver  nitrate  and  xan- 
thine silver  nitrate  have  been  formed.  The  former  is  insoluble  in 
the  cold  solution  and  separates  on  standing.  After  standing  several 
hours  filter  off  the  hypoxanthine  silver  nitrate  and  wash  with 
water  until  the  wash-water  is  only  slightly  acid  in  reaction.  Ex- 
amine the  crystals  of  hypoxanthine  silver  nitrate  under  the  micro- 
scope and  compare  them  with  those  in  Fig.  79,  page  245.  Now 
wash  the  crystals  from  the  paper  into  a  beaker  with  a  little  water 
and  warm  the  liquid.  Remove  the  silver  by  hydrogen  sulphide  and 
filter.  By  this  means  hypoxanthine  nitrate  has  been  formed  and  is 
present  in  the  filtrate.  Concentrate  on  a  water-bath  to  drive  off 
hydrogen  sulphide  and  render  the  solution  slightly  alkaline  with 
ammonia.  Warm  for  a  time,  to  remove  the  free  ammonia,  filter, 
concentrate  the  filtrate  to  a  small  volume  and  allow  it  to  stand  in 
a  cool  place.  Hypoxanthine  should  crystallize  in  small  colorless 
needles.     Examine  the  crystals  under  the  microscope. 

3.  Xanthine. — To  the  filtrate  from  the  above  experiment  con- 
taining the  xanthine  silver  nitrate  add  ammonia  in  excess.  (The 
crystalline  form  of  xanthine  silver  nitrate  is  shown  in  Fig.  80, 
p.  247.)  A  brownish-red  precipitate  of  xanthine  silver  forms. 
Treat  this  suspended  precipitate  with  hydrogen  sulphide  (do  not 
use  an  excess  of  hydrogen  sulphide),  warm  the  mixture  for  a  few 
moments  and  filter  while  hot.  Concentrate  the  filtrate  to  a  small 
volume  and  put  away  in  a  cool  place  for  crystallization  (Fig.  78, 
p.  239).  To  obtain  xanthine  in  crystalline  form  special  precautions 
are  generally  necessary.  Evaporate  the  solution  to  dryness.  Make 
the  following  tests  on  the  crystals  or  residue : 

(a)  Xanthine  Test. — Place  about  one-half  of  the  crystalline  or 
amorphous  material  in  a  small  evaporating  dish,  add  a  few  drops 
of  concentrated  nitric  acid  and  evaporate  to  dryness  very  carefully 
on  a  water-bath.  The  yellow  residue  upon  moistening  with  caustic 
potash  becomes  red  in  color  and  upon  further  heating  assumes  a 
purplish-red  hue.  Now  add  a  few  drops  of  water  and  warm. 
In  this  way  a  yellow  solution  results  which  yields  a  red  residue  upon 
evaporation.  .  How  does  this  differ  from  the  Murexide  test  upon 
uric  acid? 


MUSCULAR    TISSUE. 


247 


(b)  W ridel's  Reaction. — By  gently  heating  bring  the  remain- 
der of  the  xanthine  crystals  or  residue  into  solution  in  bromine- 
water.  Evaporate  the  solution  to  dryness  on  a  water-bath.  Re- 
move the  stopper  from  an  ammonia  bottle  and  by  blowing  across 


Fig.    8c 


Xanthine   Silver   Nitrate. 


the  mouth  of  the  bottle  direct  the  fumes  of  ammonia  so  that  they 
come  in  contact  with  the  dry  residue.  Under  these  conditions  the 
presence  of  xanthine  is  shown  by  the  residue  assuming  a  red  color. 
A  somewhat  brighter  color  may  be  obtained  by  using  a  trace  of 
nitric  acid  with  the  bromine-water.  By  the  use  of  this  modification 
however  we  may  get  a  positive  reaction  with  bodies  other  than 
xanthine. 

Hurthle's    Experiment. 

Tease  a  very  small  piece  of  frog's  muscle  on  a  microscopical  slide. 
Expose  the  slide  to  ammonia  vapor  for  a  few  moments,  then  ad- 
just a  cover  glass  and  examine  the  muscle  fibers  under  the  micro- 
scope. Note  the  large  number  of  crystals  of  ammonium  magnesium 
phosphate, 

NH4-0 

\ 

Mg-O— P  =  0 

\/ 

0 

distributed  everywhere  throughout  the  muscle  fiber,  thus  demon- 
strating the  abundance  of  phosphates  and  magnesium  in  the  muscle 
(Fig.  96,  page  301). 


CHAPTER   XVI. 

NERVOUS    TISSUE. 

In  common  with  the  other  solid  tissues  of  the  body,  nervous 
tissue  contains  a  large  amount  of  water.  The  percentage  of  water 
present  depends  upon  the  particular  form  of  nervous  tissue  but  in 
all  forms  it  is  invariably  greater  in  the  gray  matter  than  in  the 
white.  Embryonic  nervous  tissues  also  contain  a  larger  percentage 
of  water  than  the  tissues  of  adult  life.  The  gray  matter  of  the 
brain  of  the  foetus,  for  instance,  contains  about  92  per  cent  of  water, 
whereas  the  gray  matter  of  the  brain  of  the  adult  contains  but 
83-84  per  cent  of  the  fluid. 

Among  the  solid  constituents  of  nervous  tissue  are  proteins,  chol- 
esterol, cerebrin,  lecithin,  kephalin,  protagon  (f),  paranucleopro- 
tagon,  nuclein,  neurokeratin,  collagen,  extractives  and  inorganic 
salts.  The  proteins  are  present  in  the  greatest  amount  and  com- 
prise about  50  per  cent  of  the  total  solids.  Three  distinct  proteins, 
two  globulins  and  a  nucleoprotein,  have  been  isolated  from  nervous 
tissue.  The  globulins  coagulate  at  47  °  C.  and  70-75 °  C.  respec- 
tively, while  the  nucleoprotein  coagulates  at  56-600  C.  This  nuc- 
leoprotein contains  about  0.5  per  cent  of  phosphorus  (Hallibur- 
ton, Levene).  Nervous  tissue  is  composed  of  a  relatively  large 
quantity  of  a  variety  of  compounds  which  collectively  may  be 
grouped  under  the  term  "  lipoid  " — substances  resembling  the  fats 
in  some  of  their  physical  properties  and  reactions  but  distinct  in 
their  composition.  We  will  class  cerebrin,  cholesterol  and  the 
phosphorized  fats,  as  "  lipoids." 

The  group  of  phosphorized  fats  are  very  important  constituents 
of  nervous  tissue.  The  best  known  members  of  this  group  are 
lecithin,  protagon  (f)  and  kephalin.  Lecithin  occurs  in  larger 
amount  than  the  other  members  of  the  group,  has  been  more  thor- 
oughly studied  than  the  others  and  is  apparently  of  greater  impor- 
tance. Upon  decomposition  lecithin  yields  fatty  acid,  glycero-phos- 
phoric  acid  and  choline.  Each  lecithin  molecule  contains  two  fatty 
acid  radicals  which  may  be  those  of  the  same  or  different  fatty 
acids.     Thus  we  have  different  lecithins  depending  upon  the  particu- 

248 


NERVOUS    TISSUE.  249 

lar  fatty  acid  radicals  which  are  present  in  the  molecule.     The  for- 
mula of  a  typical  lecithin  would  be  the  following: 


J 


CH20-C17H35CO 
JHO  -C17H3BCO 

CH90-P0-0-C,H4 

\ 

(CH3)3-N 

/ 
OH  HO 

This  lecithin  would  be  called  distearyl-lecithin  or  choline-distearyl- 
glycero-phosphoric  acid.  Upon  decomposition  the  molecule  splits 
according  to  the  following  reaction : 

C44H90NPO9+3H2O=2(C18H3GO2)+C.,H0PO6+C5H15NO2. 

Lecithin.  Stearic  acid.  Glycero-phosphoric  Choline. 

acid. 

The  lecithins  are  not  confined  to  the  nervous  tissues  but  are  found 
in  nearly  all  animal  and  vegetable  tissues.  Lecithin  is  a  primary 
constituent  of  the  cell.  It  is  soluble  in  chloroform,  ether,  alcohol, 
benzene  and  carbon  disulphide.  The  chloroform  or  alcohol-ether 
solution  may  be  precipitated  by  acetone.  Lecithin  may  be  caused  to 
crystallize  in  the  form  of  small  plates  by  cooling  the  alcoholic  solu- 
tion to  a  low  temperature.  It  has  the  power  of  combining  with 
acids  and  bases,  and  the  hydrochloric  acid  combination  has  the  power 
of  forming  a  double  salt  with  platinic  chloride. 

Choline,  as  was  indicated  above,  is  one  of  the  decomposition 
products  of  lecithin.  It  is  trimethyl-oxyethyl-ammonhtm  hydroxide 
and  has  the  following  formula : 

CH2CH2(OH) 

/ 

N-(CH3)3 

\ 
OH 

Recent  researches  have  shown  that  great  importance  is  to  be  attached 
to  the  detection  of  choline  in  the  cerebro-spinal  fluid  and  the  blood 
in  certain  cases  of  degenerative  disease  of  the  nervous  system.  In 
this  connection  tests  for  choline  (see  p.  252)  are  of  interest  and 
value. 

Protagon,  another  nitrogenous  phosphorized  substance  is  a  body 


25O  PHYSIOLOGICAL    CHEMISTRY. 

over  which  there  has  been  much  discussion.  Upon  decomposition 
it  is  said  by  some  investigators  to  yield  cerebrin  and  the  decomposi- 
tion products  of  lecithin.  It  has  recently  been  shown  by  Posner 
and  Gies  as  well  as  by  Rosenheim  and  Tebb  that  protagon  is  a 
mixture  and  has  no  existence  as  a  chemical  individual. 

Kephalin  is  the  third  member  of  the  group  of  phosphorized  fats. 
It  is  precipitated  from  its  acetone-ether  extract  by  alcohol.  It 
contains  about  4  per  cent  of  phosphorus  and  has  been  given  the 
formula  C42H79NP013.  Kephalin  may  be  a  stage  in  lecithin  meta- 
bolism. 

Cerebrin,  a  substance  containing  nitrogen  but  no  phosphorus, 
is  an  important  constituent  of  the  white  matter  of  nervous  tissue. 
It  has  also  been  found  in  the  spleen,  pus  and  in  egg  yolk.  It  may 
be  extracted  from  the  tissue  by  boiling  alcohol  and  is  insoluble  in 
cold  alcohol,  cold  and  hot  ether  and  in  water  and  dilute  alkalis. 
Cerebrin  is  a  mixture  containing  phrenosin  (pseudo-cerebrin  or 
cerebron),  a  body  yielding  the  carbohydrate  galactose  on  decom- 
position. 

Cholesterol,  one  of  the  primary  cell  constituents,  is  present  in 
fairly  large  amount  in  nervous  tissue.  It  is  a  mon-atomic  alcohol 
with  the  formula  C27H45OH.  It  was  formerly  called  a  "non-sap- 
onifiable  fat  "  but  since  it  is  not  changed  in  any  way  by  boiling  al- 
kalis it  is  not  a  fat.  It  is  soluble  in  ether,  chloroform,  benzene  and 
hot  alcohol.  It  crystallizes  in  the  form  of  thin,  colorless,  transparent 
plates  (Fig.  42,  p.  159).  Cholesterol  occurs  abundantly  in  one  form 
of  biliary  calculus.  It  has  also  been  found  in  feces,  wool  fat,  egg 
yolk,  and  milk,  frequently  in  the  form  of  its  esters  of  higher  fatty 
acids. 

Paranucleoprotagon  is  a  phosphorized  substance  originally  iso- 
lated from  brain  tissue  by  Ulpiani  and  Lelli  and  recently  reinvesti- 
gated by  Steel  and  Gies.  It  is  said  to  possess  lecithoprotein  char- 
acteristics. 

Nervous  tissue  yields  about  1  per  cent  of  ash  which  is  made  up 
in  great  part  of  alkaline  phosphates  and  chlorides. 

Experiments   on   the   Lipoids  of   Nervous   Tissue.1 

1.  Preparation  of  Lecithin. — Treat  the  macerated  brain  of  a 
sheep  with  ether  and  allow   it  to   stand   in   the  cold   for  48-72 

1  Preparation  of  So-called  Protagon. — Macerate  the  brain  of  a  sheep,  treat 
with  85  per  cent  alcohol  and  warm  on  a  water-bath  at  450  C.  for  two  hours. 
Filter  hot  into  a  bottle  or  strong  flask  and  cool  to  0°  C.  for  one-half  hour  by 


NERVOUS    TISSUE.  25  I 

hours.  The  cold  ether  will  extract  lecithin  and  cholesterol.  Filter 
and  add  acetone  to  the  filtrate  to  precipitate  the  lecithin.  Filter 
off  the  lecithin  and  test  it  as  follows : 

(a)  Microscopical  Examination. — Suspend  a  small  portion  in  a 
drop  of  water  on  a  slide  and  examine  under  the  microscope. 

(b)  Osmic  Acid  Test. — Treat  a  small  portion  with  osmic  acid. 
What  happens? 

(c)  Acrolein  Test. — Make  the  acrolein  test  according  to  direc- 
tions on  page  135. 

(d)  "Fusion"  Test  for  Phosphorus. — Place  some  of  the  leci- 
thin prepared  above  in  a  small  porcelain  crucible,  add  a  suitable 
amount  of  a  fusion  mixture  composed  of  potassium  hydroxide  and 
potassium  nitrate  (5:1)  and  heat  carefully  until  the  resulting 
mixture  is  colorless.  Cool,  dissolve  the  mass  in  a  little  warm  water, 
acidify  with  nitric  acid,  heat  to  boiling  and  add  a  few  cubic  centi- 
meters of  molybdic  solution.  In  the  presence  of  phosphorus  a 
yellow  precipitate  forms.     What  is  it? 

2.  Preparation  of  Cholesterol. — Place  a  small  amount  of  mac- 
erated brain  tissue  under  ether  and  stir  occasionally  for  one  hour. 
Filter,  evaporate  the  filtrate  to  dryness  on  a  water-bath  and  test 
the  cholesterol  according  to  directions  given  below.  (If  it  is  de- 
sired, the  ether  extract  from  the  so-called  protagon,  or  the  ether- 
acetone  filtrate  from  the  lecithin  may  be  used  for  the  isolation  of 
cholesterol.  In  these  cases  it  is  simply  necessary  to  evaporate  the 
solution  to  dryness  on  a  water-bath.)  Upon  the  cholesterol  pre- 
pared by  either  of  the  above  methods  make  the  following  tests : 

(a)  Microscopical  Examination. — Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  in  Fig.  42,  page  159. 

(b)  Iodine -Sulphuric  Acid  Test. — Place  a  few  crystals  of  chol- 
esterol in  one  of  the  depressions  of  a  test-tablet  and  treat  with  a 
drop  of  concentrated  sulphuric  acid  and  a  drop  of  a  very  dilute  solu- 
tion of  iodine.  A  play  of  colors,  consisting  of  violet,  blue,  green 
and  red,  results. 

(c)  The  Liebcrmann-Bur chard  Test. — Dissolve  a  few  crystals 
of  cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now 
add  10  drops  of  acetic  anhydride  and  1-3  drops  of  concentrated 
sulphuric  acid.  The  solution  becomes  red,  then  blue,  and  finally 
bluish-green  in  color. 

means  of  a  freezing  mixture.  By  this  procedure  both  protagon  and  choles- 
terol are  caused  to  precipitate.  Filter  the  cold  solution  rapidly  and  treat  the 
precipitate  on  the  paper  with  ice  cold  ether  to  dissolve  out  the  cholesterol.  The 
protagon  may  now  be  redissolved  in  warm  85  per  cent  alcohol  from  which 
solution  it  will  precipitate  upon  cooling. 


252-  PHYSIOLOGICAL    CHEMISTRY. 

(d)  Salkowski's  Test. — Dissolve  a  few  crystals  of  cholesterol  in 
a  little  chloroform  and  add  an  equal  volume  of  concentrated  sul- 
phuric acid.  A  play  of  colors  from  bluish-red  to  cherry-red  and 
purple  is  noted  in  the  chloroform,  while  the  acid  assumes  a  marked 
green  fluorescence. 

(e)  Schiff's  Reaction. — To  a  little  cholesterol  in  an  evaporating 
dish  add  a  few  drops  of  Schiff's  reagent.1  Evaporate  to  dryness 
over  a  low  flame  and  observe  the  reddish-violet  residue  which 
changes  to  a  bluish-violet. 

(/)  Phosphorus. — Test  for  phosphorus  according  to  directions 
given  on  page  251.    Is  phosphorus  present  ? 

3.  Preparation  of  Cerebrin. — Treat  the  macerated  brain  tissue, 
in  a  flask,  with  95  per  cent  alcohol  and  boil  on  a  water-bath  for 
one-half  hour,  keeping  the  volume  constant  by  adding  fresh  alcohol 
as  needed.  Filter  the  solution  hot  and  stand  the  cloudy  filtrate  away 
for  twenty-four  hours.  (If  the  filtrate  is  not  cloudy  concentrate  it 
upon  the  water-bath  until  it  is  so.)  Filter  off  the  cerebrin  and 
test  it  as  follows : 

(a)  Microscopical  Examination. — Suspend  a  small  portion  in  a 
drop  of  water  on  a  slide  and  examine  under  the  microscope. 

(b)  Solubility. — Try  the  solubility  of  cerebrin  in  the  usual  sol- 
vents and  in  hot  and  cold  alcohol  and  hot  and  cold  ether. 

(c)  Phosphorus. — Test  for  phosphorus  according  to  directions 
on  page  251.    How  does  the  result  compare  with  that  on  lecithin? 

(d)  Place  a  little  cerebrin  on  platinum  foil  and  warm.  Note 
the  odor. 

(<?)  Hydrolysis  of  Cerebrin. — Place  the  remaining  cerebrin  in 
a  small  evaporating  dish,  add  equal  volumes  of  water  and  dilute 
hydrochloric  acid  and  boil  for  one  hour.  Cool,  neutralize  with 
solid  potassium  hydroxide,  filter,  and  test  with  Fehling's  solution. 
Is  there  any  reduction,  and  if  so  how  do  you  explain  it? 

4.  Tests  for  Choline. —  (a)  Rosenheim's  Periodide  Test. — Pre- 
pare an  alcoholic  extract  of  the  fluid  under  examination,  and  after 
evaporation,  apply  Rosenheim's  iodo-potassium  iodide  solution2  to 
a  little  of  the  residue.  In  a  short  time  dark  brown  plates  and 
prisms  of  choline  periodide  begin  to  form  and  may  be  detected  by 
means  of  the  microscope.  Occasionally  they  are  large  enough  to 
be  visible  to  the  naked  eye.     They  somewhat  resemble  crystals  of 

1  Schiff's  reagent  consists  of  a  mixture  of  three  volumes  of  concentrated  sul- 
phuric acid  and  one  volume  of  10  per  cent  ferric  chloride. 

3  Prepared  by  dissolving  2  grams  of  iodine  and  6  grams  of  potassium  iodide 
in   100  c.c.  of  water. 


NERVOUS    TISSUE.  253 

haemin  (see  p.  198).  If  the  slide  be  permitted  to  stand,  thus  allow- 
ing the  fluid  to  evaporate,  the  crystals  will  disappear  and  leave 
brown  oily  drops.  They  will  reappear,  however,  upon  the  addi- 
tion of  fresh  iodine  solution.  v.  Stanek  claims  that  this  choline 
compound  has  the  formula  C5H]4NOI-IR. 

(b)  Rosenheim's  Bismuth  Test. — Extract  the  fluid  under  exam- 
ination with  absolute  alcohol,  evaporate  and  re-extract  the  residue. 
Repeat  the  extraction  several  times.  Dissolve  the  final  residue  in 
2-3  c.c.  of  water  and  add  a  drop  of  Kraut's  reagent.1  Choline  is 
indicated  by  the  appearance  of  a  bright  brick-red  precipitate. 

1  Dissolve  272  grams  of  potassium  iodide  in  water  and  add  80  grams  of  bis- 
muth subnitrate  dissolved  in  200  grams  of  nitric  acid  (sp.  gr.  1.18).  Permit 
the  potassium  nitrate  to  crystallize  out,  then  filter  it  off  and  make  the  filtrate 
up  to  1  liter  with  water. 


CHAPTER   XVII. 

URINE:  GENERAL  CHARACTERISTICS  OF  NOR- 
MAL AND   PATHOLOGICAL   URINE. 

Volume. — The  volume  of  urine  excreted  by  normal  individuals 
during  any  definite  period,  fluctuates  within  very  wide  limits.  The 
average  output  for  twenty-four  hours  is  placed  by  German  writers 
between  1,500  and  2,000  c.c.  This  value  is  not  strictly  applicable 
to  conditions  in  America  however  since  it  has  been  found  that  the 
average  normal  excretion  of  the  adult  male  American  falls  within 
the  lower  values  of  1,000-1,200  c.c.  The  volume-excretion  is  in- 
fluenced greatly  by  the  diet,  particularly  by  the  ingestion  of  fluids. 
Certain  pathological  conditions  cause  the  output  of  urine  for  any 
definite  period  to  depart  very  decidedly  from  the  normal  output. 
Among  the  pathological  conditions  in  which  the  volume  of  urine 
is  increased  above  normal  are  the  following:  Diabetes  mellitus, 
diabetes  insipidus,  certain  diseases  of  the  nervous  system,  con- 
tracted kidney,  amyloid  degeneration  of  the  kidney  and  in  convales- 
cence from  acute  diseases  in  general.  Many  drugs  such  as  calomel, 
digitalis,  acetates  and  salicylates  also  increase  the  volume  of  the 
urine  excreted.  A  decrease  from  the  normal  is  observed  in  the 
following  pathological  conditions :  Acute  nephritis,  diseases  of  the 
heart  and  lungs,  fevers,  diarrhoea  and  vomiting. 

Color. — Normal  urine  ordinarily  possesses  a  yellow  tint,  the 
depth  of  the  color  being  dependent  in  part  upon  the  density  of  the 
fluid.  The  color  of  normal  urine  is  due  principally  to  a  pigment 
called  urochrome:  traces  of  hamato  porphyrin,  urobilin  and  uroery- 
thrin,  have  also  been  detected.  Under  pathological  conditions  the 
urine  is  subject  to  pronounced  variations  in  color  and  may  contain 
many  varieties  of  pigments.  Under  such  circumstances  the  urine 
may  vary  in  color  from  an  extremely  light  yellow  to  a  very  dark 
brown  or  black.  Vogel  has  constructed  a  color  chart  which  is  of 
some  value  for  purposes  of  comparison.  The  nature  and  origin 
of  the  chief  variations  in  the  urinary  color  are  set  forth  in  tabular 
form  by  Halliburton  as  follows : 

254 


URINE. 


25! 


Color. 


Cause  of  Coloration. 


Nearly  colorless. 


Dark        yellow        to 
brown-red. 


Milky. 


Orange. 


Red  or  reddish. 


Brown      to      brown- 
black. 


Greenish-yellow, 
greenish-brown, 
approaching 
black. 


Dirty  green1  or 
blue. 


Brown-yellow       to 
red-brown,     be- 
coming    blood- 
red    upon    adding 
alkalis. 


Dilution,   or  diminution   of 
normal  pigments. 


Increase  of  normal,  or  oc- 
currence of  pathological, 
pigments. 

Fat  globules. 


Pus    corpuscles. 


Excreted  drugs. 


Pathological  Condition. 

Nervous  conditions :  hy- 
druria,  diabetes  insipi- 
dus,  granular   kidney. 

\>  ute   febrile  diseases. 


Chyluria. 

Purulent     diseases     of   the 
urinary   tract. 

Santonin,    chrysophanic 

acid. 


Haematoporphyrin. 
Unchanged  haemoglobin. 

Pigments  in  food  (log- 
wood, madder,  bilberries, 
fuchsin). 


Haematin. 


Methaemoglobin. 


Melanin. 


Hydrochinon  and  catechol. 


Bile-pigments. 


A  dark-blue  scum  on  sur- 
face, with  a  blue  deposit, 
due  to  an  excess  of  indi- 
go- forming    substances. 


Haemorrhages,     or     haemo- 
globinuria. 


Small    haemorrhages. 


Methaemoglobinuria. 


Melanotic  sarcoma. 
Carbolic-acid  poisoning. 
Jaundice. 


Cholera,  typhus  ;  seen  espe- 
cially when  the  urine  is 
putrefying. 


Substances  contained  in 
senna,  rhubarb,  and  che- 
lidonium  which  are  in- 
troduced into  the  sys- 
tem. 


Transparency. — Normal  urine  is  ordinarily  perfectly  clear  and 
transparent  when  voided.  On  standing  for  a  variable  time  however, 
a  cloud  (nubecula)  consisting  principally  of  nucleoprotein  or  mu- 
coid (see  p.  320)  and  epithelial  cells  forms.  A  turbidity  due  to  the 
precipitation  of  phosphates  is  normally  noted  in  urine  passed  after  a 
hearty  meal.     The  urine  obtained  2-3  hours  after  a  meal  or  later  is 

xThis  dirty  green  or  blue  color  also  occurs  after  the  use  of  methylene-blue 
in  the  organism. 


256  PHYSIOLOGICAL    CHEMISTRY. 

ordinarily  free  from  turbidity.  Permanently  turbid  urines  ordinar- 
ily arise  from'  pathological  conditions. 

Odor. — The  odor  of  normal  urine  is  of  a  faint,  aromatic  type. 
The  bodies  to  which  this  odor  is  due  are  not  well  known,  but  it  is 
claimed  by  some  investigators  to  be  due,  at  least  in  part,  to  the 
presence  of  minute  amounts  of  certain  volatile  organic  acids. 
When  the  urine  undergoes  decomposition,  e.  g.,  in  alkaline  fermen- 
tation a  very  unpleasant  ammoniacal  odor  is  evolved.  All  urines 
are  subject  to  such  decomposition  if  allowed  to  stand  for  a  suffi- 
ciently long  time.  Under  normal  conditions  the  urine  very  often 
possesses  a  peculiar  odor  due  to  the  ingestion  of  some  certain  drug 
or  vegetable.  For  instance,  cubebs,  copaiba,  myrtol,  saffron,  tolu 
and  turpentine  each  imparts  a  somewhat  specific  odor  to  the  urine. 
After  the  ingestion  of  asparagus,  the  urine  also  possesses  a  typical 
odor. 

Frequency  of  Urination. — The  frequency  of  urination  varies 
greatly  in  different  individuals  but  in  general  is  dependent  upon  the 
amount  of  fluid  in  the  bladder.  In  pathological  conditions  an  inflam- 
matory affection  of  the  urinary  tract  or  any  disturbance  of  the  in- 
nervation of  the  bladder  will  influence  the  frequency.  Affections 
of  the  spinal  cord  which  lead  to  an  increased  irritability  of  the  blad- 
der or  a  weakening  of  the  sphincter  will  result  in  increasing  the  fre- 
quency of  urination. 

Reaction. — The  mixed  twenty-four  hour  urinary  excretion  of  a 
normal  individual  ordinarily  possesses  an  acid  reaction  to  litmus. 
This  acidity  is  now  believed  to  be  due  to  the  presence  of  various 
acidic  radicals  and  not  to  the  presence  of  sodium  di-hydrogen  phos- 
phate as  was  formerly  held  (see  Phosphates,  p.  299).  This  con- 
clusion is  reinforced  by  the  observation  that  urine  may  be  divided 
into  two  portions,  one  part  consisting  almost  entirely  of  inorganic 
matter,  including  practically  all  of  the  phosplmtes  and  having  an 
alkaline  reaction,  the  other  containing  practically  all  of  the  organic 
substances  and  no  phosphates  and  having  an  acid  reaction.  The 
acidity  imparted  to  the  urine  by  any  particular  acid  depends  entirely 
upon  the  extent  to  which  the  acid  is  dissociable,  since  it  is  the  hy- 
drogen ion  which  is  responsible  for  the  acid  reaction. 

The  composition  of  the  food  is  perhaps  the  most  important  factor 
in  determining  the  reaction  of  the  urine.  The  reaction  ordinarily 
varies  considerably  according  to  the  time  of  day  the  urine  is  passed. 
For  instance  for  a  variable  length  of  time  after  a  meal  the  urine 
may  be  neutral  or  even  alkaline  in  reaction  to  litmus,  owing  to  the 


URINE. 


257 


claim  of  the  gastric  juice  upon  the  acidic  radicals  to  further  the 
formation  of  hydrochloric  acid  for  use  in  carrying'  out  the  diges- 
tive secretory  function.  This  change  in  reaction  is  known  as  the 
alkaline  tide  and  is  common  to  perfectly  healthy  individuals.  The 
urine  may  also  become  temporarily  alkaline  in  reaction  to  litmus, 
as  the  result  of  ingesting  alkaline  carbonates  or  certain  salts  of  tar- 

Fig.    81. 


Deposit  in  Ammoniacal  Fermentation. 
a,  Acid  ammonium  urate ;  b,  ammonium  magnesium  phosphate ;  c,  bacteria. 

Fig.   82. 


■  *&  rmM 


._  b 


Redraw  tf^<* 


1     $    "h, 

CK     ^     .*      J    \ ; 


Deposit  in  Acid  Fermentation. 


a,  Fungus,  b,  amorphous  sodium  urate  ;  c,  uric  acid  ;  d,  calcium  oxalate. 

taric  and  citric  acids  which  may  be  transformed  into  carbonates 
within  the  organism.  Normal  urine  upon  standing  for  some  time 
becomes  alkaline  in  reaction  to  litmus,  owing  to  the  inception  of  al- 
kaline or  ammoniacal  fermentation  through  the  agency  of  micro- 
organisms.    This    fermentation    has   no  especial    diagnostic    value 


258 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  83. 


/nCI^ 


except  in  cases  where  the  urine  has  undergone  this  change  within 
the  organism  and  is  voided  in  the  decomposed  state.  Ammonia- 
cal  fermentation  is  ordinarily  due  to  cystitis  or  occurs  as  the  result 
of  infection  in  the  process  of  catheterization.  A  microscopical  ex- 
amination of  such  urine  (Fig.  81,  p.  257)  shows  the  presence  of 
ammonium  magnesium  phosphate  crystals,  amorphous  phosphates 
and  not  infrequently  ammonium  urate. 

Occasionally  a  urine  which  possesses  a  normal  acidity  when 
voided,  upon  standing  instead  of  undergoing  am- 
moniacal  fermentation  as  above  described  will  be- 
come still  more  strongly  acid  in  reaction.  Such  a 
phenomenon  is  termed  acid  fermentation.  Accom- 
panying this  increased  acidity  there  is  ordinarily  a 
deepening  of  the  tint  of  the  urinary  color.  Such 
urines  may  contain  acid  urates,  uric  acid,  fungi  and 
calcium  oxalate  (Fig.  82,  p.  257).  On  standing  for 
a  sufficiently  long  time  any  urine  which  exhibits 
acid  fermentation  will  ultimately  change  in  reaction, 
due  to  the  inception  of  alkaline  fermentation,  and 
will  show  the  microscopical  deposits  characteristic 
of  such  a  urine. 

Specific  Gravity. — The  specific  gravity  of  the 
urine  of  normal  individuals  varies  ordinarily  be- 
tween 1. 01 5  and  1.025.  This  value  is  subject  to 
wide  fluctuations  under  various  conditions.  For  in- 
stance following  copious  water-  or  beer-drinking  the 
specific  gravity  may  fall  to  1.003.  or  lower,  whereas 
in  cases  of  excessive  perspiration  it  may  rise  as 
high  as  1.040  or  even  higher.  Where  a  very  accu- 
rate determination  of  the  specific  gravity  is  desired 
use  is  commonly  made  of  the  pyknometer  or  of  the 
Westphal  hydrostatic  balance.  These  instruments, 
however,  are  not  suited  for  clinical  use.  The 
clinical  method  of  determining  the  specific  gravity  is  by  means 
of  a  urinometer  (Fig.  83,  p.  258).  This  affords  a  very  rapid  method 
and  at  the  same  time  is  sufficiently  accurate  for  clinical  purposes. 
The  urinometer  is  always  calibrated  for  use  at  a  specific  temperature 
and  the  observations  made  at  any  other  temperature  must  be  sub- 
jected to  a  certain  correction  to  obtain  the  true  specific  gravity.  In 
making  this  correction  one  unit  of  the  last  order  is  added  to  the 
observed  specific  gravity  for  every  three  degrees  above  the  normal 


Urinometer  and 
Cylinder. 


URINE.  259 

temperature  and  subtracted  for  every  three  degrees  below  the  nor- 
mal temperature.  For  instance,  if  in  using  a  urinometer  calibrated 
for  15°  C.  the  specific  gravity  of  a  urine  having  a  temperature  of 
21  C.  is  determined  as  1.018  it  is  necessary  to  add  to  the  observed 
specific  gravity  two  units  of  the  third  order  to  obtain  the  real  spe- 
cific gravity  of  the  urine.  Therefore  the  true  specific  gravity,  at 
1 5°  C,  of  a  urine  having  a  specific  gravity  of  [.Ol8  at  21  °  C.  is 
1. 018  -f  0.002  =  1.020. 

Pathologically,  the  specific  gravity  may  he  subjected  to  very 
wide  variations.  This  is  especially  true  in  diseases  of  the  kidneys. 
In  acute  nephritis  ordinarily  the  urine  is  concentrated  and  of  a  high 
specific  gravity,  whereas  in  chronic  nephritis  the  reverse  conditions 
are  more  apt  to  prevail.  In  fact  under  most  conditions,  whether 
physiological  or  pathological,  the  specific  gravity  of  the  urine  is  in- 
versely proportional  to  the  volume  excreted.  This  is  not  true  of 
diabetes  mellitus,  however,  where  the  volume  of  urine  is  large  and 
the  specific  gravity  is  also  high,  owing  to  the  sugar  contained  in  the 
urine. 

The  amount  of  solids  eliminated  in  the  excretion  for  twenty-four 
hours  may  be  roughly  calculated  by  means  of  Long's  coefficient, 
i.  e.,  2.6.  The  solid  content  of  1,000  c.c.  of  urine  is  obtained  by 
multiplying  the  last  two  figures  of  the  specific  gravity  observed  at 
25 °  C.  by  2.6.  To  determine  the  amount  of  solids  excreted  in 
twenty-four  hours  if  the  volume  was  1,120  c.c.  and  the  specific  grav- 
ity was  1. 018  the  calculation  would  be  as  follows: 

(a)    18X2.6  =  46.8  grams   of   solid   matter   in    1,000   c.c.   of 

urine. 

f,  N    46.8  X  1 120  . 

\o)   -—  -==  ^2.4  grams  of  solid  matter  in  1.120  c.c.  of 

1000  °        & 

urine. 

The  coefficient  of  Haser  (2.33)  which  has  been  in  use  for  years 
probably  gives  values  that  are  inaccurate  for  conditions  existing  in 
America.  This  coefficient  was  calculated  on  the  basis  of  the  specific 
gravity  determined  at  a  temperature  of  150  C. 

Freezing-Point  (Cryoscopy). — The  freezing-point  of  a  solu- 
tion depends  upon  the  total  number  of  molecules  of  solid  matter 
dissolved  in  it.  The  determination  of  the  osmotic  pressure  by 
this  method  has  recently  come  to  be  of  some  clinical  importance 
particularly  as  an  aid  in  the  diagnosis  of  kidney  disorders.  In  this 
connection  it  is  best  to  collect  the  urine  from  each  kidney  separately 
and  determine  the  freezing-point  in  the  individual  samples  so  col- 


260 


PHYSIOLOGICAL    CHEMISTRY. 


Fig 


lected.  By  this  means  considerable  aid  in  the  diagnosis  of  renal 
diseases  may  be  secured.  The  fluids  most  frequently  examined 
cryoscopically  are  the  blood  (seep.  182)  and 
the  urine.  The  freezing-point  is  denoted 
by  A.  The  value  of  A  for  normal  urine 
varies  ordinarily  between  — 1.30  and  — 2.30 
C,  the  freezing-point  of  pure  water  being 
taken  as  o°.  A  is  subject  to  very  wide 
fluctuations  under  unusual  conditions.  For 
instance  following  copious  water-  or  beer- 
drinking  A  may  have  as  high  a  value  as 
— 0.2  °  C.  whereas  on  a  diet  containing  much 
salt  and  deficient  in  fluids  the  value  of  A 
may  be  lowered  to  — 30  C.  or  even  lower. 
The  freezing-point  of  normal  blood  is  gen- 
erally about  —  0.560  C.  and  is  not  subject  to 
the  wide  variations  noted  in  the  urine,  be- 
cause of  the  tendency  of  the  organism  to 
maintain  the  normal  osmotic  pressure  of  the 
blood  under  all  conditions.  Variations  be- 
tween —  0.5 1  °  and  — 0.62  °  C.  may  be  due 
entirely  to  dietary  conditions  but  if  any 
marked  variation  is  noted  it  can,  in  most 
cases,  be  traced  to  a  disordered  kidney 
function. 

Freezing-point  determinations  may  be 
made  by  means  of  the  Beckmann-Heiden- 
hain  apparatus  (Fig.  84,  p.  260)  or  the  Zikel 
Pektoscope.  The  Beckmann  -  Heidenhain 
apparatus  consists  of  the  following  parts : 
A  strong  battery  jar  or  beaker  (C)  fur- 
nished with  a  metal  cover  which  is  provided 
with  a  circular  hole  in  its  center.  This 
strong  glass  vessel  serves  to  hold  the  freez- 
ing mixture  by  means  of  which  the  tem- 
perature of  the  fluid  under  examination  is 
lowered.  A  large  glass  tube  (B)  designed 
as  an  air-jacket,  and  formed  after  the  man- 
ner of  a  test-tube  is  introduced  through  the 
central  aperture  in  the  metal  cover  and  into  this  air-jacket  is  low- 
ered a  smaller  tube  (A)  containing  the  fluid  to  be  tested.     A  very 


Beckmann-Heidenhain 
Freezing-point  Appa- 
ratus.    (Long.) 

D,  a  delicate  thermom- 
eter ;  C,  the  containing 
jar ;  B,  the  outside  or  air 
mantle  tube ;  A,  the  tube 
in  which  the  mixture  to 
be  observed  is  placed. 
Two  stirrers  are  shown, 
one  for  the  cooling  mix- 
ture in  the  jar  and  one 
for  the  experimental  mix- 
ture. 


URINE.  26l 

delicate  thermometer  (D),  graduated  in  hundredths  of  a  degree 
is  introduced  into  the  inner  tube  and  is  held  in  place  by  means  of 
a  cork  so  that  the  mercury  bulb  is  immersed  in  the  fluid  under  ex- 
amination but  does  not  come  in  contact  with  any  glass  surface.  A 
small  platinum  wire  stirrer  serves  to  keep  the  fluid  under  examina- 
tion well  mixed  while  a  larger  stirrer  is  used  to  manipulate  the  freez- 
ing mixture.  (Rock  salt  and  ice  in  the  proportion  1  :  3  form  a  very 
satisfactory  freezing  mixture.) 

In  making  a  determination  of  the  freezing-point  of  a  fluid  by 
means  of  the  Beckmann-Heidenhain  apparatus  proceed  as  follows : 
Place  the  freezing  mixture  in  the  battery  jar  and  add  water  (if 
necessary)  to  secure  a  temperature  not  lower  than  30  C.  In- 
troduce the  fluid  to  be  tested  into  tube  A,  place  the  thermometer 
and  platinum  wire  stirrer  in  position  and  insert  the  tube  into  the 
air  jacket  which  has  previously  been  inserted  through  the  metal 
cover  of  the  battery  jar.  Manipulate  the  two  stirrers  in  order  to 
insure  an  equalization  of  temperature  and  observe  the  course  of 
the  mercury  column  of  the  thermometer  very  carefully.  The  mer- 
cury will  gradually  fall  and  this  gradual  lowering  of  the  tempera- 
ture will  be  followed  by  a  sudden  rise.  The  point  at  which  the 
mercury  rests  after  this  sudden  rise  is  the  freezing-point.  This 
rise  is  due  to  the  fact  that  previous  to  freezing,  a  fluid  is  always 
more  or  less  over  cooled  and  the  thermometer  temporarily  registers 
a  temperature  somewhat  belozv.  the  freezing-point.  As  the  fluid 
freezes  however  there  is  a  very  sudden  change  in  the  temperature 
of  the  liquid  and  this  change  is  imparted  to  the  thermometer  and 
causes  the  rise  as  indicated.  It  occasionally  occurs  that  the  fluid 
under  examination  is  very  much  over  cooled  and  does  not  freeze. 
Under  such  circumstances  a  small  piece  of  ice  is  introduced  into  it 
by  means  of  the  side  tube  noted  in  the  figure.  This  so-called 
"  inoculation  "  causes  the  fluid  to  freeze  instantaneously.  (For 
details  of  the  method  of  determining  the  freezing-point  consult 
standard  works  on  physical  or  organic  chemistry.) 

Electrical  Conductivity. — The  electrical  conductivity  of  the 
urine  is  dependent  upon  the  number  of  inorganic  molecules  or  ions 
present,  and  in  this  differs  from  the  freezing-point  which  is  de- 
pendent upon  the  total  number  of  molecules  both  inorganic  and 
organic  which  are  in  solution.  The  conductivitv  of  the  urine  has 
been  investigated  but  slightly,  and  this  very  recently,  but  from  the 
data  secured  it  seems  that  the  value  generally  falls  below  *  =  0.03. 
The  conductivity  of  blood  serum  has  been  determined  as  *  =  0.012. 


262  PHYSIOLOGICAL    CHEMISTRY. 

Up  to  the  present  time  the  determination  of  the  electrical  conduc- 
tivity of  any  of  the  fluids  of  the  body  has  been  put  to  very  slight 
clinical  use.  Experience  may  show  the  conductivity  value  to  be  a 
more  important  aid  to  diagnosis  than  it  is  now  considered,  particu- 
larly if  it  is  taken  in  connection  with  the  determination  of  the 
freezing-point.  By  a  combination  of  these  two  methods  the  por- 
tion of  the  osmotic  pressure  due  respectively  to  electrolytes  and 
non-electrolytes  may  be  determined.  For  a  discussion  of  electrical 
conductivity,  the  method  by  which  it  is  determined  and  the  princi- 
ples involved  consult  standard  works  on  physical  or  electrochemis- 
try. 

Collection  of  the  Urine  Sample. — If  any  dependable  data  are 
desired  regarding  the  quantitative  composition  of  the  urine  the  ex- 
amination of  the  mixed  excretion  for  twenty-four  hours  is  abso- 
lutely necessary.  In  collecting  the  urine  the  bladder  may  be  emp- 
tied at  a  given  hour,  say  8  A.  M.,  the  urine  discarded  and  all  the 
urine  from  that  hour  up  to  and  including  that  passed  the  next 
day  at  8  A.  M.,  saved,  thoroughly  mixed  and  a  sample  taken  for 
analysis.     Powdered   thymol, 

CH3 


\/OH 

CH3 — CH — CH3, 

is  a  very  satisfactory  preservative  since  the  excess  may  be  re- 
moved by  nitration,  if  desired,  and  any  small  amount  which  may 
go  into  solution  will  have  no  appreciable  influence  upon  the  de- 
termination of  any  of  the  urinary  constituents.  It  has  no  reducing 
power  and  so  may  safely  be  used  to  preserve  diabetic  urines.  To 
insure  the  preservation  of  the  mixed  urine  of  the  twenty-four  hour 
period  it  is  advisable  to  place  a  small  amount  of  the  thymol  powder 
:in  the  urine  receptacle  before  the  first  fraction  of  urine  is  voided. 
In  order  to  further  insure  the  preservation  of  the  urine  the  cleaned 
and  dried  urine  receptacle  may' be  rinsed  with  an  alcoholic  solution 
of  thymol  and  subsequently  thoroughly  dried  before  introducing  the 
urine. 

Toluene  is  also  used  for  the  preservation  of  urine. 

In  certain  pathological  conditions  it  is  desirable  to  collect  the 
urine  passed  during  the  day  separately  from  that  passed  during  the 


URINE.  263 

night.  When  this  is  done  the  urine  voided  between  8  A.  M.  and 
8  P.  M.  may  be  taken  as  the  day  sample  and  that  voided  between 
8  P.  M.  and  8  A.  M.  as  the  night  sample. 

The  qualitative  testing  of  urine  voided  at  random,  except  in  a 
few  specific  instances,  is  of  no  particular  value  so  far  as  giving  us 
any  accurate  knowledge  as  to  the  exact  urinary  characteristics  of 
the  individual  is  concerned.  In  the  great  majority  of  cases  the 
qualitative  as  well  as  the  quantitative  tests  should  be  made  upon 
the  mixed  excretion  for  a  twenty-four  hour  period. 


CHAPTER   XVIII 


URINE:    PHYSIOLOGICAL    CONSTITUENTS.1 

i.  Organic  Physiological  Constituents. 


Urea. 
Uric  acid. 
Creatinine. 
Creatine. 


Ethereal  sulphuric  acids 


Hippuric  acid. 
Oxalic  acid. 


Neutral  sulphur  compounds. 


Allantom. 


Aromatic  oxyacids 


Indoxyl-sulphuric  acid. 
Phenol-  and  />-cresol-sulphuric  acids. 
Pyrocatechin-sulphuric  acid. 
,  Skatoxyl-sulphuric  acid. 


'  Cystine. 

Chondroitin-sulphuric  acid. 
Thiocyanates. 
Taurine  derivatives. 
Oxyproteic  acid. 
Alloxyproteic  acid. 
Uro ferric  acid. 

Paraoxyphenyl-acetic  acid. 
Paraoxyphenyl-propionic  acid. 
Homogentisic   acid. 
Uroleucic  acid. 
Oxymandelic  acid. 
^Kynurenic  acid. 


Benzoic  acid. 
Nucleoprotein. 
Oxaluric  acid. 

1  It  is  impossible  to  make  any  absolute  classification  of  the  physiological  and 
pathological  constituents  of  the  urine.  A  substance  may  be  present  in  the 
urine  in  small  amount  physiologically  and  be  sufficiently  increased  under  cer- 
tain conditions  as  to  be  termed  a  pathological  constituent.  Therefore  it  de- 
pends, in  some  instances,  upon  the  quantity  of  a  constituent  present  whether  it 
may  be  correctly  termed  a  physiological  or  a  pathological  constituent. 

264 


URINE. 


265 


Enzymes 


Volatile   fatty  acids 

Paralactic  acid. 
Phenaceturic  acid. 

Phosphorized  compounds. 


'  Pepsin. 

Gastric  rennin. 

Amylase, 
f  Acetic  acid. 
«J  Butyric  acid. 

Formic  acid. 


Pigments 


Ptomaines  and  leucomaines. 


Purine  bases. 


j  Glycerophosphoric  acid. 
\  Phosphocarnic  acid. 

'Urochrome. 
Urobilin. 
Uroerythrin. 

Adenine. 

Guanine. 

Xanthine. 

Epiguanine. 

Episarkine. 

Hypoxanthine. 

Paraxanthine. 

Heteroxanthine. 

i-Methylxanthine. 

2.  Inorganic  Physiological  Constituents. 


Ammonia. 

Sulphates. 

Chlorides. 

Phosphates. 

Sodium  and  potassium. 

Calcium  and  magnesium. 

Carbonates. 

Iron. 

Fluorides. 

Nitrates. 

Silicates. 

Hydrogen  peroxide. 


NH2 

UREA,  C=0. 
I 

NHo 


266 


PHYSIOLOGICAL    CHEMISTRY. 


Urea  is  the  principal  end-product  of  the  metabolism  of  protein 
substances.  It  has  been  generally  believed  that  about  90  per  cent 
of  the  total  nitrogen  of  the  urine  was  present  as  urea.  Recently, 
however,  Folin  has  shown  that  the  distribution  of  the  nitrogen  of 
the  urine  among  urea  and  the  other  nitrogen-containing  bodies 
present  depends  entirely  upon  the  absolute  amount  of  the  total 
nitrogen  excreted.  He  found  that  a  decrease  in  the  total  nitrogen 
excretion  was  always  accompanied  by  a  decrease  in  the  percentage 
of  the  total  nitrogen  excreted  as  urea,  and  that  after  so  regulating 
the  diet  of  a  normal  person  as  to  cause  the  excretion  of  total  nitro- 


Fig.  85. 


Urea. 


gen  to  be  reduced  to  3-4  grams  in  24  hours,  only  about  60  per  cent 
of  this  nitrogen  appeared  in  the  urine  as  urea.  His  experiments 
also  seem  to  show  urea  to  be  the  only  one  of  the  nitrogenous  ex- 
cretions which  is  relatively  as  well  as  absolutely  decreased  as  a 
result  of  decreasing  the  amount  of  protean  metabolized.  This  same 
investigator  reports  a  hospital  case  in  which  only  14.7  per  cent  of 
the  total  nitrogen  was  present  as  urea  and  about  40  per  cent  was 
present  as  ammonia.  Morner  had  previously  reported  a  case  in 
which  but  4.4  per  cent  of  the  total  nitrogen  of  the  urine  was  present 
as  urea,  and  26.7  per  cent  was  present  as  ammonia. 

Urea  occurs  most  abundantly  in  the  urine  of  man  and  carnivora 
and  in  somewhat  smaller  amount  in  the  urine  of  herbivora;  the 
urine  of  fishes,  amphibians  and  certain  birds  also  contains  a  small 
amount  of  the  substance.  Urea  is  also  found  in  nearly  all  the  fluids 
and  in  many  of  the  tissues  and  organs  of  mammals.     The  amount 


URINE.  267 

excreted  under  normal  conditions,  by  an  adult  man  in  24  hours  is 
about  30  grams;  women  excrete  a  somewhat  smaller  amount.  The 
excretion  is  greatest  in  amount  after  a  diet  of  meat,  and  least  in 
amount  after  a  diet  consisting  of  non-nitrogenous  foods;  this  is 
due  to  the  fact  that  the  last  mentioned  diet  has  a  tendency  to  de- 
crease the  metabolism  of  the  tissue  proteins  and  thus  cause  the  out- 
put of  urea  under  these  conditions  to  fall  below  the  output  of  urea 
observed  during  starvation.  The  output  of  urea  is  also  increased 
after  copious  water-  or  beer-drinking.  This  increase  is  probably 
due  primarily  to  the  washing  out  of  the  tissues  of  the  urea  previ- 
ously formed,  but  which  had  not  been  removed  in  the  normal  proc- 
esses, and,  secondarily  to  a  stimulation  of  protein  catabolism. 

Urea  may  be  formed  in  the  organism  from  amino  acids  such  as 
leucine,  glycocoll  and  aspartic  acid :  it  may  also  be  formed 
from  ammonium  carbonate  (NH4)2C03  or  ammonium  carbamate, 
H4N-OCONH2. 

There  are  differences  of  opinion  regarding  the  transformation  of 
the  substances  just  named  into  urea  but  there  is  rather  conclusive 
evidence  that  at  least  a  part  of  the  urea  is  formed  in  the  liver ;  it  may 
be  formed  in  other  organs  or  tissues  as  well. 

Urea  crystallizes  in  long,  colorless,  four  or  six-sided,  anhydrous, 
rhombic  prisms  (Fig.  85,  p.  266),  which  melt  at  1320  C.  and  are 
soluble  in  water  or  alcohol  and  insoluble  in  ether  or  chloroform. 
If  a  crystal  of  urea  is  heated  in  a  test-tube,  it  melts  and  decomposes 
with  the  liberation  of  ammonia.     The  residue   contains   cyanuric 

COH 

/\ 

N      N 

II        I 
HOC       COH 

\// 

N 

acid,  and  biuret, 

NH2 

I 

C  =  0 

\ 
NH 

/ 
0  =  0 

I 
NIT., 


268 


PHYSIOLOGICAL    CHEMISTRY. 


The  biuret  may  be  dissolved  in  water  and  a  reddish-violet  color  ob- 
tained by  treating  the  aqueous  solution  with  cupric  sulphate  and 
potassium  hydroxide  (see  Biuret  Test,  p.  92).  Certain  hypo- 
chlorites or  hypobromites  in  alkaline  solution  have  the  power  of 
decomposing  urea  into  nitrogen,  carbon  dioxide  and  water. 
Sodium  hypobromite  brings  about  this  decomposition,  as  follows : 

CO(NH2)2  +  3NaOBr  =  3NaJBr  +  N2  +  C02  +  2H20. 

This  property  forms  the  basis  for  a  clinical  quantitative  determina- 
tion of  urea  (see  page  374). 

Urea  has  the  power  of  forming  crystalline  compounds  with 
certain  acids :  urea  nitrate  and  urea  oxalate  are  the  most  important 
of  these  compounds.  Urea  nitrate,  CO(NH2)2  -HN03,  crystal- 
lizes ill  colorless,  rhombic  or  six-sided  tiles  (Fig.  86,  below),  which 
are  easily  soluble  in  water.  Urea  oxalate,  2  •CO(NH2)2-H2C204, 
crystallizes  in  the  form  of  rhombic  or  six-sided  prisms  or  plates 
(Fig.  88,  p.  270)  :  the  oxalate  differs  from  the  nitrate  in  being 
somewhat  less  soluble  in  water. 

A  decrease  in  the  excretion  of  urea  is  observed  in  many  diseases 
in  which  the  diet  is  much  reduced  and  in  some  disorders  as  a  result 
of  alterations  in  metabolism,  e.  g.,  myxcedema,  and  in  others  as  a 
result  of  changes  in  excretion,  as  in  severe  and  advanced  kidney  dis- 


Fig.  86. 


Urea    Nitrate. 


ease.     A  pathological  increase  is  found  in  a  large  proportion  of 
diseases  which  are  associated  with  a  toxic  state. 


URINE. 


269 


Fig.   87. 


Experiments  on    Urea. 

1.  Isolation  from  the  Urine. — Place  800  c.c.  of  urine  in  a  pre- 
cipitating jar,  add  250  c.c.  of  baryta  mixture1  and  stir  thoroughly. 
Filter  off  the  precipitate  of  phosphates,  sulphates,  urates  and  hip- 
purates  and  evaporate  the  filtrate  on  a  water-bath  to  a  thick  syrup. 
This  syrup  contains  chlorides,  creatinine,  organic  salts,  pigments 
and  urea.  Extract  the  syrup  with  warm  95  per  cent  alcohol  and 
filter  again.  The  filtrate  contains  the  urea  contaminated  with  pig- 
ment. Decolorize  the  filtrate  by  boiling  with  animal  charcoal,  filter 
again  and  stand  the  filtrate  away  in  a  cold  place  for  crystallization. 
Examine  the  crystals  under  the  microscope  and  compare  them  with 
those  shown  in  Fig.  85,  page  266. 

2.  Solubility. — Test  the  solubility  of  urea,  prepared  by  yourself 
or  furnished  by  the  instructor,  in  the  ordinary 
solvents  (see  p.  23)  and  in  alcohol  and  ether. 

3.  Melting-Point. — Determine  the  melting- 
point  of  some  pure  urea  furnished  by  the 
instructor.  Proceed  as  follows  :  Into  an  ordi- 
nary melting-point  tube,  sealed  at  one  end, 
introduce  a  crystal  of  urea.  Fasten  the  tube 
to  the  bulb  of  a  thermometer  as  shown  in  Fig. 
87,  p.  269,  and  suspend  the  bulb  and  its  at- 
tached tube  in  a  small  beaker  containing  sul- 
phuric acid.  Gently  raise  the  temperature  of 
the  acid  by  means  of  a  low  flame,  stirring 
the  fluid  continually,  and  note  the  tempera- 
ture at  which  the  urea  begins  to  melt. 

4.  Crystalline  Form. — Dissolve  a  crystal 
of  pure  urea  in  a  few  drops  of  95  per  cent 
alcohol  and  place  1-2  drops  of  the  alcoholic 
solution  on  a  microscopic  slide.  Allow  the 
alcohol  to  evaporate  spontaneously,  examine 
the  crystals  under  the  microscope  and  compare 
them  with  those  reproduced  in  Fig.  85,  p.  266. 
Recrystallize  a  little  urea  from  water  in  the 
same  way  and  compare  the  crystals  with  those 
obtained  from  the  alcoholic  solution. 

5.  Formation  of  Biuret. — Place  a  small  amount  of  urea  in  a 
dry  test-tube  and  heat  carefully  in  a  low  flame.     The  urea  melts  at 

1  Baryta  mixture  consists  of  a  mixture  of  one  volume  of  a  saturated  solution 
of  Ba(NOs)2  and  two  volumes  of  a  saturated  solution  of  Ba(OH):. 


Melting-point  Tubes 
Fastened  to  Bulb  of 
Thermometer. 


270 


PHYSIOLOGICAL    CHEMISTRY. 


1320  C.  and  liberates  ammonia.  Continue  heating  until  the  fused 
mass  begins  to  solidify.  Cool  the  tube,  dissolve  the  residue  in 
dilute  potassium  hydroxide  solution  and  add  very  dilute  cupric 
sulphate  solution  (see  p.  92).  The  purplish- violet  color  is  due  to 
the  presence  of  biuret  which  has  been  formed  from  the  urea  through 
the  application  of  heat  as  indicated.     This  is  the  reaction : 


NIL 


NH2 

0  =  0 

| 

\ 

2.0  =  0    = 

NH  +  NH3 

| 

/ 

NH2 

0  =  0 

Urea. 

1 

NH2 

Biuret. 

6.  Urea  Nitrate. — Prepare  a  concentrated  solution  of  urea  by 
dissolving  a  little  of  the  substance  in  a  few  drops  of  water.  Place 
a  drop  of  this  solution  on  a  microscopic  slide,  add  a  drop  of  con- 


FlG. 


Urea  Oxalate. 


centrated  nitric  acid  and  examine  under  the  microscope.     Compare 
the  crystals  with  those  reproduced  in  Fig.  86,  p.  268. 

7.  Urea  Oxalate. — To  a  drop  of  a  concentrated  solution  of  urea, 
prepared  as  described  in  the  last  experiment  (6),  add  a  drop  of  a 
saturated  solution  of  oxalic  acid.  Examine  under  the  microscope 
and  compare  the  crystals  with  those  shown  in  Fig.  88,  above. 


URINE.  271 

8.  Decomposition  by  Sodium-Hypobromite. —  Into  a  mixture 
of  3  c.c.  of  concentrated  sodium  hydroxide  solution  and  2  c.c.  of 

bromine  water  in  a  test-tube  introduce  a  crystal  of  urea  or  a  -.mall 
amount  of  a  concentrated  solution  of  urea.  Through  the  influence 
of  the  sodium-hypobromitc,  NaOBr,  the  urea  is  decomposed  and 
carbon  dioxide  and  nitrogen  are  liberated.  The  carbon  dioxide  is 
absorbed  by  the  excess  of  sodium  hydroxide  while  the  nitrogen  is 
evolved  and  causes  the  marked  effervescence  observed.  This 
property  forms  the  basis  for  one  of  the  methods  in  common  use  for 
the  quantitative  determination  of  urea.  Write  the  equation  show- 
ing the  decomposition  of  urea  by  sodium-hypobromite. 

9.  Furfurol  Test. — To  a  few  crystals  of  urea  in  a  small  por- 
celain dish  add  1-2  drops  of  a  concentrated  aqueous  solution  of 
furfurol  and  1-2  drops  of  a  concentrated  hydrochloric  acid.  Note 
the  appearance  of  a  yellow  color  which  gradually  changes  into 
a  purple.    Allantoin  also  responds  to  this  test  (see  page  287). 

HN-C 

uric  acid,   OC     C— NH 

'^CO. 

HN-C-NH 

Uric  acid  is  one  of  the  most  important  of  the  constituents  of  the 
urine.  It  is  generally  stated  that  normally  about  0.7  gram  is  ex- 
creted in  24  hours  but  that  this  amount  is  subject  to  wide  variations, 
particularly  under  certain  dietary  and  pathological  conditions. 
Very  recently  it  has  been  shown  that  the  average  daily  excretion  of 
uric  acid  for  ten  men  ranging  in  age  from  19  to  29  years  and  fed  a 
normal  mixed  diet  was  0.597  gram,  a  value  somewhat  lower  than 
the  generally  accepted  average  of  0.7  gram  for  such  a  period. 
Uric  acid  is  a  diureide  and  consequently  upon  oxidation  yields  two 
molecules  of  urea.  It  acts  as  a  weak  dibasic  acid  and  forms  two 
classes  of  salts,  neutral  and  acid.  The  neutral  potassium  and  lith- 
ium urates  are  the  most  easily  soluble  of  the  alkali  salts;  the  am- 
monium urate  is  difficultly  soluble.  The  acid-alkali  urates  are  more 
insoluble  and  form  the  major  portion  of  the  sediment  which  sepa- 
rates upon  cooling  concentrated  urine ;  the  alkaline  earth  urates  are 
very  insoluble.  Ordinarily  uric  acid  occurs  in  the  urine  in  the  form 
of  urates  and  upon  acidifying  the  liquid  the  uric  acid  is  liberated 
and  deposits  in  crystalline  form.     This  property  forms  the  basis 


272  PHYSIOLOGICAL    CHEMISTRY. 

of  one  of  the  older  methods  for  the  quantitative  determination  of 
uric  acid  (Heintz  Method,  p.  373). 

Uric  acid  is  very  closely  related  to  the  purine  bases  as  may  be 
seen  from  a  comparison  of  its  structural  formula  with  those  of  the 
purine  bases  given  on  page  241.  According  to  the  purine  nomen- 
clature it  is  designated  2-6— 8-trioxypurine.  Uric  acid  forms  the 
principal  end-product  of  the  nitrogenous  metabolism  of  birds  and 
scaly  amphibians;  in  the  human  organism  it  occupies  the  fourth 
position  inasmuch  as  here  urea,  ammonia  and  creatinine  are  the 
chief  end-products  of  nitrogenous  metabolism.  It  is  generally  said 
that  the  relation  existing  between  uric  acid  and  urea  in  human  urine 
under  normal  conditions  varies  on  the  average  from  1  -.40  to  1  :ioo 
and  is  subject  to  wider  variations  under  pathological  conditions; 
and  further  that  because  of  the  high  content  of  uric  acid  in  the  urine 
of  new-born  infants  the  ratio  may  be  reduced  to  1  :io  or  even 
lower.  We  now  know  that  this  ratio  of  uric  acid  to  urea  is  of 
little  significance  under  any  conditions. 

In  man,  uric  acid  probably  results  principally  from  the  destruc- 
tion of  nuclein  material.  It  may  arise  from  nuclein  or  other  purine 
material  ingested  as  food  ©r  from  the  disintegrating  cellular  matter 
of  the  organism.  The  uric  acid  resulting  from  the  first  process  is  said 
to  be  of  exogenous  origin,  whereas  the  product  of  the  second  form 
of  activity  is  said  to  be  of  endogenous  origin.  As  a  result  of  experi- 
mentation, Siven,  and  Burian  and  Schur,  and  Rockwood  claim  that 
the  amount  of  endogenous  uric  acid  formed  in  any  given  period 
is  fairly  constant  for  each  individual  under  normal  conditions,  and 
that  it  is  entirely  independent  of  the  total  amount  of  nitrogen  elimi- 
nated. Recently  Folin  has  taken  exception  to  the  statements  of 
these  investigators  and  claims  that,  following  a  pronounced  decrease 
in  the  amount  of  protein  metabolized,  the  absolute  quantity  of  uric 
acid  is  decreased  but  that  this  decrease  is  relatively  smaller  than  the 
decrease  in  the  total  nitrogen  excretion  and  that  the  per  cent  of  the 
uric  acid  nitrogen,  in  terms  of  the  total  nitrogen,  is  therefore  de- 
cidedly increased. 

In  birds  and  scaly  amphibians  the  formation  of  uric  acid  is  anal- 
ogous to  the  formation  of  urea  in  man.  In  these  organisms  it  is 
derived  principally  from  the  protein  material  of  the  tissues  and  the 
food  and  is  formed  through  a  process  of  synthesis  which  occurs  for 
the  most  part  in  the  liver;  a  comparatively  small  fraction  of  the 
total  uric  acid  excretion  of  birds  and  scaly  amphibians  may  result 
from  nuclein  material. 


PLATE  V. 


Uric  Acid  Crystals.     Normal  Color.     (From  Purdy,  after  Peyer.) 


URINE.  2/3 

When  pure,  uric  acid  may  be  obtained  as  a  white,  odorless,  and 
tasteless  powder  which  is  composed  principally  of  small  transparent 
crystalline  rhombic  plates.  Uric  acid  as  it  separates  from  the  urine 
is  invariably  pigmented,  and  crystallizes  in  a  large  variety  of  char- 
acteristic tonus,  e.  g.}  dumb-bells,  wedges,  rhombic  prisms,  irregu- 
lar rectangular  or  hexagonal  plates,  whetstones,  prismatic  rosettes, 
etc.  Uric  acid  is  insoluble  in  alcohol  and  ether,  soluble  with  diffi- 
cult}' in  boiling'  water  (1:1800)  and  practically  insoluble  in  cold 
water  (1:39,480,  at  18°  C).  It  is  soluble  in  alkalis,  alkali  car- 
bonates, boiling  glycerol,  concentrated  sulphuric  acid  and  in  cer- 
tain organic  bases  such  as  ethylamine  and  piperidine.  It  is  claimed 
that  the  uric  acid  is  held  in  solution  in  the  urine  by  the  urea  and 
disodium  hydrogen  phosphate  present.  L'ric  acid  possesses  the 
power  of  reducing  cupric  hydroxide  in  alkaline  solution  and  may 
thus  lead  to  an  erroneous  conclusion  in  testing  for  sugar  in  the  urine 
by  means  of  Fehling's  or  Trommer's  tests.  A  white  precipitate  of 
cuprous  urate  is  formed  if  only  a  small  amount  of  cupric  hydroxide 
is  present,  but  if  enough  of  the  copper  salt  is  present  the  character- 
istic red  or  brownish-red  precipitate  of  cuprous  oxide  is  obtained. 
Uric  acid  does  not  possess  the  power  of  reducing  bismuth  in  alka- 
line solution  and  therefore  does  not  interfere  in  testing  for  sugar 
in  the  urine  by  means  of  Boettger's  or  Nylander's  tests. 

In  addition  to  being  an  important  urinary  constituent  uric  acid  is 
normally  present  in  the  brain,  heart,  liver,  lungs,  pancreas  and 
spleen ;  it  also  occurs  in  the  blood  of  birds  and  has  been  detected  in 
traces  in  human  blood  under  normal  conditions. 

Pathologically,  the  excretion  of  uric  acid  is  subject  to  wide  vari- 
ations but  the  experimental  findings  are  rather  contradictorv.  It 
may  be  stated  with  certainty,  however,  that  in  leukaemia  the  uric 
acid  output  is  increased  absolutely  as  well  as  relatively  to  the  urea 
output;  under  these  conditions  the  ratio  between  the  uric  acid  and 
urea  may  be  as  low  as  1:9,  whereas  the  normal  ratio,  as  we  have 
seen,  is  1:50  or  higher.  In  the  study  of  the  influence  of 
X-ray  on  metabolism  Edsall  has  very  recently  reached  some  in- 
teresting conclusions.  He  found  that  the  excretion  of  uric  acid  is 
usually  increased  and  that  in  some  conditions,  particularly  in 
leukaemia,  it  may  be  greatly  increased.  The  excretion  of  total 
nitrogen,  phosphates  and  other  substances  may  also  be  considerably 
increased. 

!9 


274 


PHYSIOLOGICAL    CHEMISTRY, 


Experiments  on  Uric  Acid. 

i.  Isolation  from  the  Urine. — Place  about  200  c.c.  of  filtered 
urine  in  a  beaker,  render  it  acid  with  2-10  c.c.  of  concentrated 
hydrochloric  acid,  stir  thoroughly  and  stand  the  vessel  in  a  cold 
place  for  24  hours.  Examine  the  pigmented  crystals  of  uric  acid 
under  the  microscope  and  compare  them  with  those  shown  in  Fig. 
101,  p.  347  and  PI.  V.,  opposite  p.  273. 

2.  Solubility. — Try  the  solubility  of  pure  uric  acid,  furnished  by 
the  instructor,  in  the  ordinary  solvents  (see  p.  23)  and  in  alcohol, 
ether,  concentrated  sulphuric  acid  and  in  boiling  glycerol. 

3.  Crystalline  Form  of  Pure  Uric  Acid. — Place  about  100  c.c. 
of  water  in  a  small  beaker,  render  it  distinctly  alkaline  with  potas- 
sium hydroxide  solution  and  add  a  small  amount  of  pure  uric  acid, 
stirring  continuously.  Cool  the  solution,  render  it  distinctly  acid 
with  hydrochloric  acid  and  allow  it  to  stand  in  a  cool  place  for  crys- 
tallization. Examine  the  crystals  under  the  microscope  and  com- 
pare them  with  those  reproduced  in  Fig.  89,  below. 

4.  Murexide  Test. — To  a  small  amount  of  pure  uric  acid  in  a 
small  evaporating  dish  add  2-3  drops  of  concentrated  nitric  acid. 
Evaporate  to  dryness  carefully  on  a  water-bath  or  over  a  very  low 

Fig.  8g. 


Pure    Uric    Acid. 


flame.  A  red  or  yellow  residue  remains  which  turns  purplish-red 
after  cooling  the  dish  and  adding  a  drop  of  very  dilute  ammonium 
hydroxide.  The  color  is  due  to  the  formation  of  murexide.  If  po- 
tassium hydroxide  is  used  instead  of  ammonium  hydroxide  a  pur- 


URINE.  2/5 

plish-violet  color  clue  to  the  production  of  the  potassium  sail  is  ob- 
tained. The  color  disappears  upon  warming;  with  certain  related 
bodies  (purine  bases)  the  color  persists  under  these  conditions. 

5.  Moreigne's  Reaction. — To  equal  volumes  of  Moreigne's  re- 
agent' and  the  solution  to  be  tested  add  a  tew  drops  of  concen- 
trated potassium  hydroxide.  A  blue  color  indicates  the  presence  of 
uric  acid. 

6.  Schiff's  Reaction. — Dissolve  a  small  amount  of  pure  uric  acid 
in  sodium  carbonate  solution  and  transfer  a  drop  of  the  resulting 
mixture  to  a  strip  of  filter  paper  saturated  with  argentic  nitrate 
solution.  A  yellowish-brown  or  black  coloration  due  to  the  forma- 
tion of  reduced  silver  is  produced. 

7.  Influence  upon  Fehling's  Solution. — Dilute  1  c.c.  of  Feh- 
ling's  solution  with  4  c.c.  of  water  and  heat  to  boiling.  Now  add 
slozvly,  a  few  drops  at  a  time,  1-2  c.c.  of  a  concentrated  solution  of 
uric  acid  in  potassium  hydroxide,  heating  after  each  addition. 
From  this  experiment  what  do  you  conclude  regarding  the  possi- 
bility of  arriving'  at  an  erroneous  decision  when  testing  for  sugar 
in  the  urine  by  means  of  Fehling's  test? 

8.  Reduction  of  Nylander's  Reagent. — To  5  c.c.  of  a  solution 
of  uric  acid  in  potassium  hydroxide  add  about  one-half  a  cubic 
centimeter  of  Nylander's  reag"ent  and  heat  to  boiling  for  a  few  mo- 
ments. Do  you  obtain  the  typical  black  end-reaction  signifying  the 
reduction  of  the  bismuth  ? 

NH CO 

I 

CREATININE,     C  =  NH 

N  •  CH3  •  CH2. 

Creatinine  is  the  anhydride  of  creatine  and  is  a  constituent  of 
normal  human  urine.  The  theory  that  creatinine  is  derived  from 
the  creatine  of  ingested  muscular  tissue  as  well  as  from  the  creatine 
of  the  muscular  tissue  of  the  organism  has  recently  been  proven 
to  be  incorrect  by  Folin,  Klercker,  and  \\o\i  and  Shaffer.  Shaffer 
believes  that  creatinine  is  the  result  of  some  special  process 
of  normal  metabolism  which  takes  place  to  a  large  extent,  if  not 
entirely,  in  the  muscles  and  further  that  the  amount  of  such  creatin- 

1  Moreigne's  reagent  is  made  by  combining  20  grams  of  sodium  tungstate,  10 
grams  of  phosphoric  acid  (sp.  gr.  1.13)  and  100  c.c.  of  water.  Boil  this  mixture 
for  twenty  minutes,  add  water  to  make  the  volume  of  the  solution  equivalent 
to  the  original  volume  and  acidify  with  hydrochloric  acid. 


276  PHYSIOLOGICAL    CHEMISTRY. 

ine  elimination,  expressed  in  milligrams  per  kilogram  body  weight, 
is  an  index  of  this  special  process.1  He  further  states  that  the  mus- 
cular efficiency  of  the  individual  depends  upon  the  intensity  of  this 
process.  Under  normal  conditions  about  1  gram  of  creatinine  is  ex- 
creted by  an  adult  man  in  24  hours,2  the  exact  amount  depending 
in  great  part  upon  the  nature  of  the  food  and  decreasing  markedly 
in  starvation.  Very  little  that  is  important  is  known  regarding 
the  excretion  of  creatinine  under  pathological  conditions.  The 
creatinine  content  of  the  urine  is  said  to  be  increased  in  typhoid 
fever,  typhus,  tetanus  and  pneumonia,  and  to  be  decreased  in  ansemia, 
chlorosis,  paralysis,  muscular  atrophy,  advanced  degeneration  of 
the  kidneys,  and  in  leucaemia  (myelogenous,  lymphatic  and 
pseudo).  An  increase  of  creatinine  was  also  noted  in  diabetes, 
an  increase  probably  due  to  the  creatinine  content  of  the 
meat  eaten.  The  greater  part  of  the  data,  however,  relating  to  the 
variation  of  the  creatinine  excretion  under  pathological  conditions 
are  not  of  much  value  since  in  nearly  every  instance,  the  diet  was  not 
sufficiently  controlled  to  permit  the  collection  of  reliable  data.  And 
further,  until  the  advent  of  the  Folin  method  (see  p.  392),  there 
was  no  accurate  method  for  the  quantitative  determination  of  cre- 
atinine. Shaffer  has  very  recently  called  attention  to  the  fact  that 
a  low  excretion  of  creatinine  is  found  in  the  urine  of  a  remarkably 
large  number  of  pathological  subjects,  representing  a  variety  of  con- 
ditions, and  that  it  is  therefore  evident  that  the  excretion  of  an  ab- 
normally small-amount  of  this  substance  is  by  no  means  peculiar  to 
any  one  disease. 

Creatinine  crystallizes  in  colorless,  glistening  monoclinic  prisms 
(Fig.  90,  p.  277)  which  are  soluble  in  about  12  parts  of  cold  water; 
they  are  more  soluble  in  warm  water  and  in  warm  alcohol.  One 
of  the  most  important  and  interesting  of  the  compounds  of  creatin- 
ine is  creatinine -zinc  chloride,  (C4H7N30)2ZnCl2,  which  is  formed 
from  an  alcoholic  solution  of  creatinine  upon  treatment  with  zinc 
chloride  in  acid  solution.  Creatinine  has  the  power  of  reducing 
cupric  hydroxide  in  alkaline  solution  and  in  this  way  may  interfere 
with  the  determination  of  sugar  in  the  urine.  In  the  reduction  by 
creatinine  the  blue  liquid  is  first  changed  to  a  yellow  and  the  for- 
mation of  a  brownish-red  precipitate  of  cuprous  oxide  is  brought 

1  He  proposes  to  designate  as  the  "  creatinine  coefficient "  the  excretion  of 
creatinine-nitrogen   (mgs.)   per  kilogram  of  body  weight. 

'  According  to  Shaffer  the  amount  excreted  by  strictly  normal  individuals 
is  between  7  and  11  milligrams  of  creatinine-nitrogen  per  kilogram  of  body 
weight. 


URINE.  277 

about  only  after  continuous  boiling  w  ith  an  excess  of  the  copper  salt. 
Creatinine  does  not  reduce  alkaline  bismuth  solutions  and  there  tore 
does  not  interfere  with  Xvlander's  and  Boettger's  tests. 

Fig.  <)o. 


Creatinine. 

It  has  recently  been  shown  by  Folin  that  the  absolute  quantity  of 
creatinine  eliminated  in  the  urine  on  a  meat-free  diet  is  a  constant 
quantity  different  for  different  individuals,  but  wholly  independ- 
ent of  quantitative  changes  in  the  total  amount  of  nitrogen  elimin- 
ated. Shaffer  has  very  recently  confirmed  these  findings  and  has 
shown  that  the  output  of  creatinine  under  these  conditions  is  con- 
stant from  hour  to  hour  as  well  as  from  day  to  day. 

Experiments   on    Creatinine. 

1.  Separation  from  the  Urine. — Place  250  c.c.  of  urine  in  a 
casserole  or  beaker,  render  it  alkaline  with  milk  of  lime  and  then 
add  CaCl2  solution  until  the  phosphates  are  completely  precipitated. 
Filter  off  the  precipitate,  render  the  filtrate  slightly  acid  with  acetic 
acid  and  evaporate  it  to  a  syrup.  While  still  warm  this  syrup  is 
treated  with  about  50  c.c.  of  95-  97  per  cent  alcohol  and  the  mix- 
ture allowed  to  stand  8-12  hours  in  a  cool  place.  The  precipitate 
is  now  filtered  off  and  the  filtrate  treated  with  a  little  sodium  acetate 
and  about  one-half  c.c.  of  acid-free  zinc  chloride  solution  having  a 
specific  gravity  of  1.2.  This  mixture  is  stirred  thoroughly  and 
allowed  to  stand  in  a  cold  place  for  48-72  hours.  Creatinine- 
zinc  chloride  (Fig.  91,  p.  278)  will  crystallize  out  under  these  con- 


278 


PHYSIOLOGICAL    CHEMISTRY. 


ditions.  Collect. the  crystals  on  a  filter  paper  and  wash  them  with 
alcohol  to  remove  chlorides.  Now  treat  the  zinc  chloride  compound 
with  a  little  warm  water,  boil  with  lead  oxide  and  filter.  The  fil- 
trate may  now  be  decolorized  by  animal  charcoal,  evaporated  to 


Fig.  91. 


Creatinine-Zinc   Chloride.      (Salkowski.) 


dryness  and  the  residue  extracted  with  strong-  alcohol.  (Creatine 
remains  undissolved  under  these  conditions. )  The  alcoholic  extract 
of  creatinine  is  now  evaporated  to  incipient  crystallization  and  left 
in  a  cool  place  until  crystallization  is  complete.  If  desired  the  crys- 
tals may  be  purified  by  recrystallization  from  water. 

2.  Weyl's  Test. — Take  5  c.c.  of  urine  in  a  test-tube,  add  a  few 
drops  of  sodium  nitro-prusside  and  render  the  solution  alkaline 
with  potassium  hydroxide  solution.  A  ruby-red  color  results  which 
soon  turns  yellow.     See  Legal's  test  for  acetone,  page  329. 

3.  Salkowski's  Test. — To  the  yellow  solution  obtained  in  Weyl's 
test  above  add  an  excess  of  acetic  acid  and  apply  heat.  A  green 
color  results  and  is  in  turn  displaced  by  a  blue  color.  A  precipi- 
tate of  Prussian  blue  may  form. 

4.  Jaffe's  Reaction. — Place  5  c.c.  of  urine  in  a  test-tube,  add  an 
aqueous  solution  of  picric  acid  and  render  the  mixture  alkaline  with 
potassium  hydroxide  solution.  A  red  color  is  produced  which  turns 
yellow  if  the  solution  be  acidified.  Dextrose  gives  a  similar  red 
color  but  only  upon  the  application  of  heat.  This  color  reaction 
observed  when  creatinine  in  alkaline  solution  is  treated  with  picric 
acid  is  the  basic  principle  of  Folin's  colorimetric  method  for  the 
quantitative  determination  of  creatinine  (see  page  392). 


URINE.  279 

ETHEREAL    SULPHURIC    ACIDS. 

The  most  important  of  the  ethereal  sulphuric  acids  found  in  the 
urine  are  phenol-sulphuric  acid,  p-cresol-^sulphuric  acid,  indoxyl- 
sulphuric  acid  and  skatoxyl-sulphuric  acid,  l'yrocatechin-sulphuric 
acid  also  occurs  in  traces  in  human  urine.  The  total  output  of 
ethereal  sulphuric  acid  varies  from  0.09  to  0.62  gram  for  24  hours. 
In  health  the  ratio  of  ethereal  sulphuric  acid  to  inorganic  sulphuric 
acid  is  about  1  :  10.  These  ethereal  sulphuric  acids  originate  in  part 
from  the  phenol,  cresol,  indole  and  skatole  formed  in  the  putrefac- 
tion of  protein  material  in  the  intestine.  The  phenol  passes  to  the 
liver  where  it  is  conjugated  to  form  phenyl  potassium  sulphate  and 
appears  in  this  form  in  the  urine  whereas  the  indole  and  skatole 
undergo  a  preliminary  oxidation  to  form  indoxyl  and  skatoxyl 
respectively  before  their  elimination. 

It  has  generally  been  considered  that  each  of  the  ethereal  sul- 
phuric acids  was  formed  principally  in  the  putrefaction  of  protein 
material  in  the  intestine  and  that  therefore  a  determination  of  the 
total  ethereal  sulphuric  acid  content  of  the  urine  was  an  index  of  the 
extent  to  which  these  putrefactive  processes  were  proceeding  within 
the  organism.  Recently,  however,  Folin  has  conducted  a  series  of 
experiments  which  seem  to  show  that  the  ethereal  sulphuric  acid  con- 
tent of  the  urine  does  not  afford  an  index  of  the  extent  of  intestinal 
putrefaction,  since  these  bodies  arise  only  in  part  from  putrefactive 
processes.  He  claims  that  the  ethereal  sulphuric  acid  excretion 
represents  a  form  of  sulphur  metabolism  which  is  more  in  evidence 
upon  a  diet  containing  a  very  small  amount  of  protein  or  upon  a 
diet  containing  absolutely  no  protein.  The  ethereal  sulphuric 
acid  content  of  the  urine  diminishes  as  the  total  sulphur 
content  diminishes  but  the  percentage  decrease  is  much  less.  There- 
fore when  considered  from  the  standpoint  of  the  total  sulphuric 
acid  content  the  ethereal  sulphuric  acid  content  is  not  diminished 
but  is  increased,  although  the  total  sulphuric  acid  content  is  dimin- 
ished. Folin's  experiments  also  seem  to  show  that  the  indoxyl 
sulphuric  acid  (indoxyl  potassium  sulphate  or  indican )  content  of 
the  urine  does  not  originate  to  any  degree  from  the  metabolism 
of  protein  material  but  that  it  arises  in  great  part  from  intestinal 
putrefaction  and  that  the  excretion  of  indoxyl  sulphuric  acid  may 
alone  be  taken  as  a  rough  index  of  the  extent  of  putrefactive  proc- 
esses within  the   intestine.     Indoxyl   sulphuric  acid, 


28o  PHYSIOLOGICAL    CHEMISTRY. 

CH 

//\ 
HC      C-C(0-S08H), 

I        II      II 
HC      C     CH 

\/\/ 
CH  NH 

therefore,  which  occurs  in  the  urine  as  indoxyl  potassium  sulphate 
or  indican, 

CH 

//\ 

HC      C-C(0-S03K), 

I        II      II 
HC      C     CH 

\/\/ 
CH  NH 

is  clinically  the  most  important  of  the  ethereal  sulphuric  acids. 

Tests    for    Indican. 

i.  Jaffe's  Test. — Nearly  fill  a  test-tube  with  a  mixture  composed 
of  equal  volumes  of  concentrated  HC1  and  the  urine  under  exam- 
ination. Add  2-3  c.c.  of  chloroform  and  a  few  drops  of  a  calcium 
hypochlorite  solution,  place  the  thumb  over  the  end  of  the  test- 
tube  and  shake  the  tube  and  contents  thoroughly.  The  chloroform 
is  colored  more  or  less,  according  to  the  amount  of  indican  present. 
Ordinarily  a  blue  color  due  to  the  formation  of  indigo-blue  is 
produced ;  less  frequently  a  red  color  due  to  indigo-red  may  be  noted. 

This  is  the  reaction  (see  also  pages  162  and  163)  : 

CH 

//\ 
HC      C-C-OH 

2      I        ||        II  +20  = 

HC      C      CH 

CH   NH 

Indoxyl,  C8H7NO. 

CH  CH 

//\  /\ 

HC      O-C-0  O-C-C      CH 

I         I        II  I        II        I       +2H20 

HC      C      C  C      C      CH 

V  \/  \/  \// 

CH    NH  NH    CH 

Indigo-blue,  Ci6Hi0N2O2. 


URINE.  251 

2.  Obermayer's  Test. — Nearly  fill  a  test-tube  with  a  mixture 
composed  of  equal  volumes  of  Obermayer's  reagent1  and  the  urine 
under  examination.     Add  2-3  c.c.  of  chloroform,  place  the  thumb 

over  the  end  of  the  test-tube  and  shake  thoroughly.      How    does 
this  compare  with  Jaffe's  test? 

3.  Giirber's  Reaction. — To  one  volume  of  the  urine  under  ex- 
amination and  two  volumes  of  concentrated  hydrochloric  acid  in  a 
test-tube  add  2-3  drops  of  a  1  per  cent  solution  of  osmic  acid  and 
2-3  c.c.  of  chloroform  and  shake  the  tube  and  contents  thoroughly. 
Compare  the  color  with  those  obtained  in  Jaffe's  and  Obermayer's 
tests. 

An  excess  of  osmic  acid  does  not  affect  the  reaction.  Occa- 
sionally better  results  are  obtained  if  the  solution  of  osmic  acid 
is  added  directly  to  the  urine  before  the  addition  of  the  hydro- 
chloric acid.  If  the  urine  under  examination  be  strongly  colored 
or  of  high  specific  gravity  it  should  first  be  treated  with  basic  lead 
acetate  (one-eighth  volume).  The  precipitate  is  then  removed  by 
filtration  and  the  resulting  filtrate  used  in  making  the  test  for 
indican. 

4.  Rossi's  Reaction. — To  equal  volumes  of  concentrated  hydro- 
chloric acid  and  the  urine  under  examination,  in  a  test-tube,  add 
1  drop  of  a  10  per  cent  solution  of  ammonium  persulphate  and  2-7, 
c.c.  of  chloroform.  Agitate  the  mixture  vigorously  and  note  the 
color  of  the  chloroform.  Compare  this  result  with  those  obtained 
in  the  other  indican  tests. 

5.  Lavelle's  Reaction. — To  10  c.c.  of  urine  in  a  test-tube  add 
2-3  c.c.  of  Obermayer's  reagent1  and  a  similar  volume  of  concen- 
trated sulphuric  acid.  (During  the  addition  of  the  acid  the  tube 
should  be  held  under  running  water  in  order  that  the  temperature 
of  the  mixture  may  not  rise  too  high.)  Add  2-7,  c.c.  of  chloro- 
form, shake  the  tube  vigorously,  and  observe  the  depth  of  color 
assumed  by  the  chloroform. 

The  sponsor  for  this  reaction  claims  it  to  be  the  most  satisfactory 
of  the  indican  tests. 

CO  XHCH2COOH. 

HIPPURIC  ACID, 

\/ 

'Obermayer's  reagent  is  prepared  by  adding  J-4  grams  ,it"  tonic  chloride  to 
a  liter  of  concentrated  HC1   (sp.  gr.   1.10). 


282 


PHYSIOLOGICAL    CHEMISTRY. 


This  acid  occurs  normally  in  the  urine  of  both  the  carnivora  and 
herbivora  but  is  more  abundant  in  the  urine  of  the  latter.  It  is 
formed  by  a  synthesis  of  benzoic  acid  and  glycocoll  which  takes 
place  in  the  kidneys.  The  average  excretion  of  an  adult  man 
for  24  hours  under  normal  conditions  is  about  0.7  gram.     Hippuric 


Hippuric   Acid. 

acid  crystallizes  in  needles  or  rhombic  prisms  (see  Fig.  92,  above) 
the  particular  form  depending  upon  the  rapidity  of  crystallization. 
Pure  hippuric  acid  melts  at  1870  C.  The  most  satisfactory  method 
for  the  isolation  of  hippuric  acid  from  the  urine  in  crystalline 
form  is  that  proposed  by  Roaf  (see  p.  283).  It  is  easily  soluble 
in  alcohol  or  hot  water,  and  only  slightly  soluble  in  ether.  The 
output  of  hippuric  acid  is  increased  in  diabetes  owing  probably  to  the 
ingestion  of  much  protein  and  fruit.  It  is  decreased  in  fevers 
and  in  certain  kidney  disorders  where  the  synthetic  activity  of  the 
renal  cells  is  diminished.  Hippuric  acid  may  be  determined  quan- 
titatively by  means  of  Dakin's  methods  (see  p.  383). 


Experiments   on    Hippuric   Acid. 

1.  Separation  from  the  Urine,  (a)  First  Method. — Render  500- 
1000  c.c.  of  urine  of  the  horse  or  cow1  alkaline  with  milk  of  lime, 

1  If  urine  of  the  horse  or  cow  is  not  available  human  urine  may  serve  the 
purpose  fully  as  well  provided  means  are  taken  to  increase  it's  content  of  hippuric 
acid.  This  may  be  conveniently  accomplished  by  ingesting  2  grams  of  ammonium 
benzoate  at  night.  The  fraction  of  urine  passed  in  the  morning  will  be  found 
to  have  a  high  content  of  hippuric  acid.  The  ammonium  benzoate  is  in  no  way 
harmful. 


URINE.  283 

boil  for  a  few  moments  and  filter  while  hot.  Concentrate  the  fil- 
trate, over  a  burner,  to  a  small  volume.  Cool  the  solution,  acidify 
it  strongly  with  concentrated  hydrochloric  acid  and  stand  it  in  a 
cool  place  for  24  hours.  Filter  off  the  crystals  of  hippuric  acid 
which  have  formed  and  wash  them  with  a  little  cold  water.  Re- 
move the  crystals  from  the  paper,  dissolve  them  in  a  very  small 
amount  of  hot  water  and  percolate  the  hot  solution  through  thor- 
oughly washed  animal  charcoal,  being  careful  to  wash  out  the  last 
portion  of  the  hippuric  acid  solution  with  hot  water.  Filter,  con- 
centrate the  filtrate  to  a  small  volume  and  stand  it  aside  for  crys- 
tallization. Examine  the  crystals  under  the  microscope  and  com- 
pare them  with  those  in  Fig.  92,  page  282.  This  method  is  not  as 
satisfactory  as  Roaf's  method  (see  below). 

(b)  Roaf's  Method. — Place  500  c.c.  of  urine  of  the  horse  or 
cow1  in  a  casserole  or  precipitating  jar  and  add  an  equal  volume 
of  a  saturated  solution  of  ammonium  sulphate2  and  7.5  c.c.  of  con- 
centrated sulphuric  acid.  Permit  the  mixture  to  stand  for  twenty- 
four  hours  and  remove  the  crystals  of  hippuric  acid  by  filtration. 
Purify  the  crystals  by  recrystallization  according  to  the  directions 
given  above  under  First  Method.  Examine  the  crystals  under  the 
microscope  and  compare  them  with  those  given  in  Fig.  92,  p.  282. 

If  sufficient  urine  is  not  available  to  permit  the  use  of  500  c.c. 
a  smaller  volume  may  be  used  inasmuch  as  it  is  possible,  by  the 
above  technique,  to  isolate  hippuric  acid  in  crystalline  form  from  as 
small  a  volume  as  25-50  c.c.  of  herbivorous  urine.  The  greater 
the  amount  of  ammonium  sulphate  added  the  more  rapid  the 
crystallization  until  at  the  saturation  point  the  crystals  of  hippuric 
acid  sometimes  form  in  about  ten  minutes. 

2.  Melting-Point. — Determine  the  melting-point  of  the  hip- 
puric acid  prepared  in  the  above  experiment  (see  p.  269). 

3.  Solubility. — Test  the  solubility  of  hippuric  acid  in  the  ordi- 
nary solvents  (page  23)  and  in  alcohol,  and  ether. 

4.  Dehn's  Reaction.  —  Introduce  about  5  c.c.  of  the  urine  or  the 
solution  under  examination  into  a  test-tube  and  add  sufficient  hvpo- 
bromite  solution3  to  impart  to  the  mixture  a  permanent  yellow  color. 
In  the  case  of  urine  enough  hypobromite  should  be  added  to  decom- 
pose the  urea.  Fleat  the  mixture  to  boiling  and  note  the  forma- 
tion of  an  orange  or  brown-red  precipitate  if  hippuric  acid  is  pres- 

1  See  note  on  p.  282. 

2 125  grams  of  solid  ammonium  sulphate  may  be  substituted. 

3  See  note  on  p.  375. 


284  PHYSIOLOGICAL    CHEMISTRY. 

ent.  If  the  solution  under  examination  contains  only  a  trace  of 
hippuric  acid  the  solution  will  appear  smoky  and  faintly  red  in  color 
whereas  if  a  larger  amount  of  the  acid  be  present  the  solution 
will  become  opaque  and  of  an  orange  or  brown-red  color.  In  either 
case  after  standing  for  some  time  the  solution  should  clear  up  and 
a  light,  finely  divided  precipitate  should  be  deposited.  This  precip- 
itate consists  of  earthy  phosphates  mixed  with  an  amorphous  orange 
or  brown-red  substance  of  unknown  composition. 

5.  Formation  of  Nitro-Benzene. — To  a  little  hippuric  acid  in 
a  small  porcelain  dish  add  1-2  c.c.  of  concentrated  HNOs  and 
evaporate  to  dryness  on  a  water-bath.  Transfer  the  residue  to  a 
dry  test-tube,  apply  heat  and  note  the  odor  of  the  artificial  oil  of 
bitter  almonds  (nitro-benzene). 

6.  Sublimation. — Place  a  few  crystals  of  hippuric  acid  in  a  dry 
test-tube  and  apply  heat.  The  crystals  are  reduced  to  an  oily  fluid 
which  solidifies  in  a  crystalline  mass  upon  cooling.  When  stronger 
heat  is  applied  the  liquid  assumes  a  red  color  and  finally  yields  a 
sublimate  of  benzoic  acid  and  the  odor  of  hydrocyanic  acid. 

7.  Formation  of  Ferric  Salt. — Render  a  small  amount  of  a  so- 
lution of  hippuric  acid  neutral  with  dilute  potassium  hydroxide. 
Now  add  1-3  drops  of  neutral  ferric  chloride  solution  and  note  the 
formation  of  the  ferric  salt  of  hippuric  acid  as  a  cream  colored 
precipitate. 

COOH 

OXALIC  ACID,     J 

COOH. 

Oxalic  acid  is  a  constituent  of  normal  urine,  about  0.02  gram 
being  eliminated  in  24  hours.  It  is  present  in  the  urine  as  cal- 
cium oxalate,  which  is  kept  in  solution  through  the  medium  of  the 
acid  phosphates.  The  origin  of  the  oxalic  acid  content  of  the  urine 
is  not  well  understood.  It  is  eliminated,  at  least  in  part,  unchanged 
when  ingested,  therefore  since  many  of  the  common  articles  of 
diet,  e.  g.,  asparagus,  apples,  cabbage,  grapes,  lettuce,  spinach, 
tomatoes,  etc.,  contain  oxalic  acid  it  seems  probable  that  the  in- 
gested food  supplies  a  portion  of  the  oxalic  acid  found  in  the  urine. 
There  is  also  experimental  evidence  that  part  of  the  oxalic  acid 
of  the  urine  is  formed  within  the  organism  in  the  course  of  protein 
and  fat  metabolism.  It  has  also  been  suggested  that  oxalic  acid 
may  arise  from  an  incomplete  combustion  of  carbohydrates,  espe- 
cially under  certain   abnormal  conditions.     Pathologically,   oxalic 


URINE.  2.S5 

acid  is  found  to  be  increased  in  amount  in  diabetes  mellitUS,  in  or- 
ganic diseases  of  the  liver  and  in  various  other  conditions  which 
are  accompanied  by  a  derangement  of  the  oxidation  mechanism. 
An  abnormal  increase  of  oxalic  acid  is  termed  oxaluria.  A  consid- 
erable increase  in  the  content  ot  oxalic  acid  may  be  noted  unaccom- 
panied by  any  other  apparent  symptom.  Calcium  oxalate  crystal- 
lizes in  at  least  two  distinct  forms,  dumb-bells  and  octahedra  I  Fig. 

99..  Pa§"e  345)- 

Experiments. 

Preparation  of  Calcium  Oxalate.  First  Method. — Place  200- 
250  c.c.  of  urine  in  a  beaker,  add  5  c.c.  of  a  saturated  solution  of 
calcium  chloride,  make  the  urine  slightly  acid  with  acetic  acid  and 
stand  the  beaker  aside  in  a  cool  place  for  24  hours.  Examine  the 
sediment  under  the  microscope  and  compare  the  crystalline  forms 
with  those  shown  in  Fig.  99,  p.  345. 

Second  Method. — Proceed  as  above,  replacing  the  acetic  acid  by 
an  excess  of  ammonium  hydroxide  and  filtering  off  the  precipitate 
of  phosphates. 

NEUTRAL    SULPHUR   COMPOUNDS. 

Under  this  head  may  be  classed  such  bodies  as  cystine  (see  p. 
72),  chrondroitin-sulphuric  acid,  oxyproteic  acid,  alloxyproteic  acid, 
uroferric  acid,  thiocyanates  and  taurine  derivatives.  The  sul- 
phur content  of  the  bodies  just  enumerated  is  generally  termed 
loosely  combined  or  neutral  sulphur  in  order  that  it  may  not  be  con- 
fused with  the  acid  sulphur  which  occurs  in  the  inorganic  sulphuric 
acid  and  ethereal  sulphuric  acid  forms.  Ordinarily  the  neutral  sul- 
phur content  of  normal  human  urine  is  14-20  per  cent  of  the  total 
sulphur  content. 

NHCHHN 


ALLANTOIN,   00 


00. 


NH-CO  NH2 

Allantoi'n  has  been  found  in  the  urine  of  suckling  calves  as  well 
as  in  that  of  the  dog  and  cat.  It  has  also  been  detected  in  the  urine 
of  infants  within  the  first  eight  days  after  birth,  as  well  as  in 
the  urine  of  adults.  It  is  more  abundant  in  the  urine  of  women 
during"  pregnancy.  Underhil]  also  reports  the  presence  of  allantoin 
in  the  urine  of  fasting  dog's,  an  observation  which  makes  it  probable 


280  PHYSIOLOGICAL    CHEMISTRY. 

that  allantoin  is  a  constant  constituent  of  the  urine  of  such  animals. 
Allantoin  is  formed  by  the  oxidation  of  uric  acid  and  the  output  is 
increased  by  thymus  or  pancreas  feeding.  When  pure  it  crystal- 
lizes in  prisms  (Fig.  93,  below)  and  when  impure  in  granules  and 

Fig.  93. 


Allantoin,    from    Cat's   Urine. 

a  and  b,  Forms  in  which  it  crystallized  from  the  urine ;  c,  re-crystallized  allantoin. 
(Drawn  from  micro-photographs  furnished  by  Prof.  Lafayette  B.  Mendel  of  Yale 
University.) 

knobs.       Pathologically,  it  has  been  found  increased  in  diabetes 
insipidus  and  in  hysteria  with  convulsions    (Pouchet). 

Experiments. 

1.  Separation  from  the  Urine.1 — Meissner's  Method. — Precipi- 
tate the  urine  with  baryta  water.  Neutralize  the  nitrate  carefully 
with  dilute  sulphuric  acid,  filter  immediately  and  evaporate  the  fil- 
trate to  incipient  crystallization.  Completely  precipitate  this  warm 
fluid  with  95  per  cent  alcohol  (reserve  the  precipitate).  Decant  or 
filter  and  precipitate  the  solution  by  ether.  Combine  the  ether  and 
alcohol  precipitates  and  extract  with  cold  water  or  hot  alcohol; 
allantoin  remains  undissolved.  Bring  the  allantoin  into  solution  in 
hot  water  and  recrystallize. 

Allantoin  may  be  determined  quantitatively  by  the  Paduschka- 
Underhill-Kleiner  method  (see  p.  407)  or  by  Loewi's  method.2 

2.  Preparation  from   Uric   Acid. — Dissolve  4  grams   of  uric 

1  The  urine  of  the  dog  after  thymus,  pancreas  or  uric  acid  feeding  may  be 
employed. 
2Archiv  fur  Experimentelle  Pathologie  und  Pharmakologie,   1900,  xliv,  p.  20. 


URINE.  287 

acid  in  too  c.c.  of  water  rendered  alkaline  with  potassium  hydrox- 
ide.    COol  nnd  carefully  add  3  grams  of  potassium  permanganate. 

Filter,  immediately  acidulate  the  filtrate  with  acetic  acid  and  allow 
it  to  stand  in  a  cool  place  over  night.  Filter  off  the  crystal-  and 
wash  them  with  water.  Save  the  wash  water  and  filtrate,  unite 
them  and  after  concentrating  to  a  small  volume  stand  away  for 
crystallization.  Now  combine  all  the  crystals  and  recrystallize  them 
from  hot  water.  Use  these  crystals  in  the  experiments  which 
follow. 

3.  Microscopical  Examination. — Examine  the  crystals  made 
in  the  last  experiment  and  compare  them  with  those  shown  in  Fig. 
93,  page  286. 

4.  Solubility. — Test  the  solubility  of  allantoin  in  the  ordinary 
solvents  (page  23). 

5.  Reaction. — Dissolve  a  crystal  in  water  and  test  the  reaction 
to  litmus. 

6.  Furfurol  Test. — Place  a  few  crystals  of  allantoin  on  a  test- 
tablet  or  in  a  porcelain  dish  and  add  1-2  drops  of  a  concentrated 
aqueous  solution  of  furfurol  and  1-2  drops  of  concentrated  hydro- 
chloric acid.  Observe  the  formation  of  a  yellow  color  which  turns 
to  a  light  purple  if  allowed  to  stand.  This  test  is  given  by  urea 
but  not  by  uric  acid. 

7.  Murexide  Test. — Try  this  test  according  to  the  directions 
given  on  page  274.     Note  that  allantoin  fails  to  respond. 

8.  Reduction  of  Fehling's  Solution. — Make  this  test  in  the 
usual  way  (see  p.  27)  except  that  the  boiling  must  be  prolonged 
and  excessive.  Ultimately  the  allantoin  will  reduce  the  solution. 
Compare  with  the  result  on  uric  acid,  page  275. 

AROMATIC   OXYACIDS. 

Two  of  the  most  important  of  the  oxyacids  are  paraoxy-phenyl- 
acetic  acid, 

CHoCOOH, 

0 

OH 

and  paraoxy phenyl-propionic  acid, 

CH2CH2COOH. 


OH 


288  PHYSIOLOGICAL    CHEMISTRY. 

They  are  products  of  the  putrefaction  of  protein  material  and 
tyrosine  is  an  intermediate  stage  in  their  formation.  Both  these 
acids  for  the  most  part  pass  unchanged  into  the  urine  where  they 
occur  normally  in  very  small  amount.  The  content  may  be  in- 
creased in  the  same  manner  as  the  phenol  content,  in  particular  by 
acute  phosphorus  poisoning.  A  fraction  of  the  total  aromatic  oxy- 
acid  content  of  the  urine  is  in  combination  with  sulphuric  acid,  but 
the  greater  part  is  present  in  the  form  of  salts  of  sodium  and 
potassium. 

Homogentisic  Acid  or  di-oxyphenyl-acetic  acid, 

OH 
j^iCH2-COOH, 

\y 

OH 

is  another  important  oxyacid  sometimes  present  in  the  urine. 
Under  the  name  glycosuric  acid  it  was  first  isolated  from  the  urine 
by  Prof.  John  Marshall  of  the  University  of  Pennsylvania;  sub- 
sequently Baumann  isolated  it  and  determined  its  chemical  constitu- 
tion. It  occurs  in  cases  of  alcaptonuria.  A  urine  containing  this 
oxyacid  turns  greenish-brown  from  the  surface  downward  when 
treated  with  a  little  sodium  hydroxide  or  ammonia.  If  the  solution 
be  stirred  the  color  very  soon  becomes  dark  brown  or  even  black. 
Homogentisic  acid  reduces  alkaline  copper  solutions  but  not  alka- 
line bismuth -solutions.  Uroleucic  acid  is  similar  in  its  reactions 
to  homogentisic  acid. 

Oxyiiiandelic  Acid  or  paraoxyphenyl-glycolic  acid, 

OH 


CH(OH)COOH, 

has  been  detected  in  the  urine  in  cases  of  yellow  atrophy  of  the 
liver. 

Kynurenic  Acid  or  y-oxy-/?-quinoline  carbonic  acid, 

CH     COH 

HC        C        C-COOH, 

I         II  I 

HC        C        CH 

CH     N 


I -RINK. 


289 


is  present  in  the  urine  of  the  dog  and  has  recently  been  detected 
by  Swain  in  the  urine  of  the  coyote.  To  isolate  it  from  the  urine 
proceed  as  follows:  Acidify  the  urine  with  hydrochloric  aeid  in 
the  proportion  1  :  .25.  From  this  aeid  fluid  both  the  uric  aeid  and 
the  kynurenic  aeid  separate  in  the  course  of  24  48  hours.  Filter 
off  the  combined  crystalline  deposit  of  the  two  acids,  dissolve  the 
kynurenic  acid  in  dilute  ammonia  (uric  acid  is  insoluble)  and  re- 
precipitate  it   with   hydrochloric  acid. 

Kynurenic  acid   may  he  quantitatively  determined   by   Capaldi's 
method.1 

COOH. 


BENZOIC  ACID, 


\, 


Benzoic  acid  has  been  detected  in  the  urine  of  the  rabbit  and  dog. 
It  is  also  said  to  occur  in  human  urine  accompanying  renal  disor- 
ders. The  benzoic  acid  probably  originates'  from  a  fermentative 
decomposition  of  the  hippuric  acid  of  the  urine. 


Experiments. 

1.  Solubility. — Test  the  solubility  of  benzoic  acid  in  water,  alco- 
hol and  ether. 

Fig.  94. 


Benzoic    Acid. 

2.  Crystalline  Form. — Recrystallize  some  benzoic  acid  from  hot 
water,  examine  the  crystals  under  the  microscope  and  compare 
them  with  those  reproduced  in  Fig.  94.  above. 

1  Zeitschrift  fiir  physiologische  Chemie,  1897,  xxiii,  p.  92. 

20 


29O  PHYSIOLOGICAL    CHEMISTRY. 

3.  Sublimation. — Place  a  little  benzoic  acid  in  a  test-tube  and 
heat  over  a  flame.  Note  the  odor  which  is  evolved  and  observe 
that  the  acid  sublimes  in  the  form  of  needles. 

4.  Dissolve  a  little  sodium  benzoate  in  water  and  add  a  solution 
of  neutral  ferric  chloride.  Note  the  production  of  a  brownish-yel- 
low precipitate  (salicylic  acid  gives  a  reddish-violet  color  under  the 
same  conditions).  Add  ammonium  hydroxide  to  some  of  the  pre- 
cipitate. It  dissolves  and  ferric  hydroxide  is  formed.  Add  a  little 
hydrochloric  acid  to  another  portion  of  the  original  precipitate  and 
stand  the  vessel  away  over  night.     What  do  you  observe? 

NUCLEOPROTEIN. 

The  nubecula  of  normal  urine  has  been  shown  by  one  investigator 
to  consist  of  a  mucoid  containing  12.7  per  cent  of  nitrogen  and 
2.3  per  cent  of  sulphur.  This  body  evidently  originates  in  the  uri- 
nary passages.  It  is  probably  slightly  soluble  in  the  urine.  Some 
investigators  believe  that  the  body  forming  the  nubecula  of  normal 
urine  is  nucleoprotein  and  not  a  mucin  or  mucoid  as  stated  above. 
A  discussion  of  nucleoprotein  and  related  bodies  occurring  in  the 
urine  under  pathological  conditions  will  be  found  on  page  320. 

NH-CO 

OXALURIC  ACID,   CO 

NH2    COOH. 

Oxaluric  acid  is  not  a  constant  constituent  of  normal  human 
urine,  and  when  found  occurs  only  in  traces  as  the  ammonium 
salt.     Upon  boiling  oxaluric  acid  it  splits  into  oxalic  acid  and  urea. 

ENZYMES. 

Various  types  of  enzymes  produced  within  the  organism  are  ex- 
creted in  both  the  feces  and  the  urine.  In  this  connection  it  is 
interesting  to  note  that  pepsin,  gastric  rennin  and  an  amylase  have 
been  positively  identified  in  the  urine.  The  occurrence  of  trypsin 
in  the  urine,  at  least  under  normal  conditions,  is  questioned. 

VOLATILE  FATTY  ACIDS. 

■  Acetic,  butyric  and  formic  acids  have  been  found  under  normal 
conditions  in  the  urine  of  man  and  of  certain  carnivora  as  well  as 
in  the  urine  of   herbivora.     Normally  they  arise  principally  from 


URINE.  29I 

the  fermentation  of  carbohydrates  and  the  putrefaction  of  proteins. 

The  acids  containing"  the  fewest  carbon  atoms  I  formic  and  acetic) 
are  found  to  be  present  in  larger  percentage  than  those  which  con 
tain  a  larger  number  of  such  atoms.  The  volatile  fatty  acid-  occur 
in  normal  urine  in  traces,  the  total  output  tor  twenty-four  hour-, 
according  to  different  investigators,  varying  from  0.008  gram  to 
0.05  gram. 

Pathologically,  the  excretion  of  volatile  fatty  acids  is  increased 
in  diabetes,  fevers,  and  in  certain  hepatic  diseases  in  which  the 
parenchyma  of  the  liver  is  seriously  affected.  Under  other  patho- 
logical conditions  the  output  may  be  diminished.  These  variations, 
however,  in  the  excretion  of  the  volatile  fatty  acids  possess  very 
little  diagnostic  value. 

CH3 

PARALACTIC    ACID,    OH  (OH) 

I 

COOH. 

Paralactic  acid  is  supposed  to  pass  into  the  urine  when  the  supply 
of  oxygen  in  the  organism  is  diminished  through  any  cause,  c.  g., 
after  acute  yellow  atrophy  of  the  liver,  acute  phosphorus  poisoning 
or  epileptic  attacks.  This  acid  has  also  been  found  in  the  urine  of 
healthy  persons  following  the  physical  exercise  incident  to  pro- 
longed marching.  Paralactic  acid  has  been  detected  in  the  urine 
of  birds  after  the  removal  of  the  liver.  Underhill  reports  the  oc- 
currence of  this  acid  in  the  urine  of  a  case  of  pernicious  vomiting 
of  pregnancy. 

CH.COXHCHoCOOH. 
/\ 

PHENACETURIC  ACID,   I 


Phenaceturic  acid  occurs  principally  in  the  urine  of  herbivorous 
animals  but  has  frequently  been  detected  in  human  urine.  It  is  pro- 
duced in  the  organism  through  the  synthesis  of  glycocoll  and 
phenylacetic  acid.  It  may  be  decomposed  into  its  component  parts 
by  boiling  with  dilute  mineral  acids.  The  crystalline  form  of 
phenaceturic  acid  (small  rhombic  plates  with  rounded  angles)  re- 
sembles one  form  of  uric  acid  crystal. 

PHOSPHORIZED  COMPOUNDS. 

Phosphorus  in  organic  combination  has  been  found  in  the  urrne 
in  such  bodies  as  glycerophosphoric  acid,  which   may   arise    from 


292  PHYSIOLOGICAL    CHEMISTRY. 

the  decomposition  of  lecithin,  and  phosphocarnic  acid.  It  is  claimed 
that  on  the  average  about  2.5  per  cent  of  the  total  phosphorus  elimi- 
nation is  in  organic  combination. 

PIGMENTS. 

There  are  at  least  three  pigments  normally  present  in  human 
urine.     These  pigments  are  urochrome,  urobilin  and  uroerythrin. 

A.     UROCHROME. 

This  is  the  principal  pigment  of  normal  urine  and  imparts  the 
characteristic  yellow  color  to  that  fluid.  It  is  apparently  closely 
related  to  its  associated  pigment  urobilin  since  the  latter  may  be 
readily  converted  into  urochrome  through  evaporation  of  its  aque- 
ous-ether solution.  Urochrome  may  be  obtained  in  the  form  of  a 
brown,  amorphous  powder  which  is  readily  soluble  in  water  and 
95  per  cent  alcohol.  It  is  less  soluble  in  absolute  alcohol,  acetone, 
amyl  alcohol  and  acetic  ether  and  insoluble  in  benzene,  chloroform 
and  ether.  Urochrome  is  said  to  be  a  nitrogenous  body  (4.2  per 
cent  nitrogen),  free  from  iron. 

B.     UROBILIN. 

Urobilin,  which  was  at  one  time  considered  to  be  the  principal 
pigment  of  urine,  in  reality  contributes  little  toward  the  pigmenta- 
tion of  this  fluid.  It  is  claimed  that  no  urobilin  is  present  in 
freshly  voided  normal  urine  but  that  its  precursor,  a  chromogen 
called  urobilinogen,  is  present  and  gives  rise  to  urobilin  upon  de- 
composition through  the  influence  of  light.  It  is  claimed  by  some 
investigators  that  there  are  various  forms  of  urobilin,  e.  g.,  normal, 
febrile,  physiological  and  pathological.  Urobilin  is  said  to  be  very 
similar  to,  if  not  absolutely  identical  with,  hydrobilirubin  (see 
page  173). 

Urobilin  may  be  obtained  as  an  amorphous  powder  which  varies 
in  color  from  brown  to  reddish-brown,  red  and  reddish-yellow  de- 
pending upon  the  way  in  which  it  is  prepared.  It  is  easily  soluble  in 
ethyl  alcohol,  amyl  alcohol  and  chloroform,,  and  slightly  soluble  in 
ether,  acetic  ether  and  in  water.  Its  solutions  show  characteristic 
absorption-bands  (see  Absorption  Spectra,  Plate  II).  Under 
normal  conditions  urobilin  is  derived  from  the  bile  pigments  in  the 
intestine. 

Urobilin  is  increased  in  most  acute  infectious  diseases  such  as  ery- 
sipelas, malaria,  pneumonia  and  scarlet  fever.     It  is  also  increased 


URINE.  293 

in  appendicitis,  carcinoma  of  the  liver,  catarrhal  icterus,  pernicious 
ainciuia  and  in  cases  of  poisoning  by  antifebrin,  antipyrin,  pyridin, 
and  potassium  chlorate.  In  general  if  is  usually  increased  when 
blood  destruction  is  excessive  and  in  many  disturbances  of  the  liver. 
It  is  markedly  decreased  in  phosphorus  poisoning. 

Experiments. 

1.  Spectroscopic  Examination. — Acidify  the  urine  with  hydro- 
chloric acid  and  allow  it  to  remain  exposed  to  the  air  for  a  few- 
moments.  By  this  means  if  any  urobilinogen  is  present  it  will  be 
transformed  into  urobilin.  The  urine  may  now  be  examined  by 
means  of  the  spectroscope.  If  urobilin  is  present  in  the  fluid  the 
characteristic  absorption-band  lying"  between  b  and  F  will  be  ob- 
served (see  Absorption  Spectra,  Plate  II).  It  may  be  found  neces- 
sary to  dilute  the  urine  with  water  before  a  distinct  absorption- 
band  is  observed.  This  test  may  be  modified  by  acidifying  10  c.c. 
of  urine  with  hydrochloric  acid  and  shaking-  it  gently  with  5  c.c.  of 
amyl  alcohol.  The  alcoholic  extract  when  examined  spectro- 
scopicallv  will  show  the  characteristic  urobilin  absorption-band. 
(Note  the  spectroscopic  examination  in  the  next  experiment.  ) 

2.  Ammoniacal-Zinc  Chloride  Test. — Render  some  of  the  urine 
ammoniacal  by  the  addition  of  ammonium  hydroxide,  and  after 
allowing"  it  to  stand  a  short  time  filter  off  the  precipitate  of  phos- 
phates and  add  a  few  drops  of  zinc  chloride  solution  to  the  filtrate. 
Observe  the  production  of  a  greenish  fluorescence.  Examine  the 
fluid  by  means  of  the  spectroscope  and  note  the  absorption-band 
which  occupies  much  the  same  position  as  the  absorption-band  of 
urobilin  in  acid  solution  (see  Absorption  Spectra.  Plate  II). 

3.  Gerhardt's  Test. — To  20  c.c.  of  urine  add  3-5  c.c.  of  chloro- 
form and  shake  well.  Separate  the  chloroform  extract  and  add  to 
it  a  few  drops  of  iodine  solution  (I  in  KI).  Render  the  mixture 
alkaline  with  dilute  solution  of  potassium  hydroxide  and  note  the 
production  of  a  yellow  or  yellowish-brown  color.  The  solution 
ordinarily  exhibits  a  greenish  fluorescence. 

4.  Wirsing's  Test. — To  20  c.c.  of  urine  add  3  5  c.c.  of  chloro- 
form and  shake  gently.  Separate  the  chloroform  extract  and  add 
to  it  a  drop  of  an  alcoholic  solution  of  zinc  chloride.  Note  the 
rose-red  color  and  the  greenish  fluorescence.  If  the  solution  is 
turbid  it  may  be  rendered  clear  by  the  addition  of  a  tew  c.c.  of 
absolute  alcohol. 

c.  Ether-Absolute  Alcohol  Test. — Mix  urine  and  pure  ether 


294  PHYSIOLOGICAL    CHEMISTRY. 

in  equal  volumes  and  shake  gently  in  a  separatory  funnel.  Sepa- 
rate the  ether  extract,  evaporate  it  to  dryness  and  dissolve  the  resi- 
due in  2-3  c.c.  of  absolute  alcohol.  Note  the  greenish  fluorescence. 
Examine  the  solution  spectroscopically  and  observe  the  characteristic 
absorption-band    (see  Absorption  Spectra,   Plate  II). 

6.  Ring  Test. — Acidify  25  c.c.  of  urine  with  2-3  drops  of  con- 
centrated hydrochloric  acid,  add  5  c.c.  of  chloroform  and  shake  the 
mixture.  Separate  the  chloroform,  place  it  in  a  test-tube  and  add 
carefully  3-5  c.c.  of  an  alcoholic  solution  of  zinc  acetate.  Observe 
the  formation  of  a  green  ring  at  the  zone  of  contact  of  the  two 
fluids.     If  the  tube  is  shaken  a  fluorescence  may  be  observed. 

C.     UROERYTHRIN. 

This  pigment  is  frequently  present  in  small  amount  in  normal 
urine.  The  red  color  of  urinary  sediments  is  due  in  great  part  to 
the  presence  of  uroerythrin.  It  is  easily  soluble  in  amyl  alcohol, 
slightly  soluble  in  acetic  ether,  absolute  alcohol  or  chloroform,  and 
nearly  insoluble  in  water.  Dilute  solutions  of  uroerythrin  are  pink 
in  color  while  concentrated  solutions  are  orange-red  or  bright  red : 
none  of  its  solutions  fluoresce.  Uroerythrin  is  increased  in  amount 
after  strenuous  physical  exercise,  digestive  disturbances,  fevers,  cer- 
tain liver  disorders  and  in  various  other  pathological  conditions. 

PTOMAINES   AND   LEUCOMAINES. 

These  toxic  substances  are  said  to  be  present  in  small  amount  in 
normal  urine.  Very  little  is  known,  definitely,  however,  about 
them.  It  is  claimed  that  five  different  poisons  may  be  detected  in  the 
urine,  and  it  is  further  stated  that  each  of  these  substances  pro- 
duces a  specific  and  definite  symptom  when  injected  intravenously 
into  a  rabbit.  The  resulting  symptoms  are  narcosis,  salivation, 
mydriasis,  paralysis  and  convulsions.  The  day  urine  is  principally 
narcotic  and  is  2-4  times  as  toxic  as  the  night  urine  which  is  chiefly 
productive  of  convulsions. 

PURINE  BASES. 

The  purine  bases  found  in  human  urine  are  adenine,  carnine, 
epiguanine,  episarkine,  guanine,  xanthine,  heteroxanthine,  hypo- 
xanthine,  paraxanthine  and  i-methylxanthine.  The  main  bulk  of 
the  purine  base  content  of  the  urine  is  made  up  of  paraxanthine. 
heteroxanthine  and  i-methylxanthine  which  are  derived  for  the 
most  part  from  the  caffeine,  theobromine  and  theophylline  of  the 


URINE.  295 

food.  The  total  purine  base  content  is  made  up  of  the-  products 
of  two  distinct  forms  of  metabolism,  i.  <•.,  metabolism  of  ingested 
nucleins  and  purines  and  metabolism  of  tissue  nuclein  material. 
Purine  bases  resulting  from  the  fust  Form  of  metabolism  are  said 
to  be  of  exogenous  origin  whereas  those  resulting  from  the  second 
form  of  metabolism  are  said  to  be  of  endogenous  origin.  The  daily 
output  of  purine  bases  by  the  urine  is  extremely  small  and  varies 
greatly  with  the  individual  (16  60  milligrams).  The  output  is  in- 
creased after  the  ingestion  of  nuclein  material  as  well  as  after  the 
increased  destruction  of  leucocytes.  A  well  marked  increase  ac- 
companies leukaemia.  Edsall  has  very  recently  shown  that  the  out- 
put of  purine  bases  by  the  urine  is  increased  as  a  result  of  X-ray 
treatment. 

Experiment. 

1.  Formation  of  the  Silver  Salts. — Add  an  excess  of  magnesia 
mixture1  to  25  c.c.  of  urine.  Filter  off  the  precipitate  and  add  am- 
moniacal  silver  solution2  to  the  filtrate.  A  precipitate  composed  of 
the  silver  salts  of  the  various  purine  bases  is  produced.  The  purine 
bases  may  be  determined  quantitatively  by  means  of  Kriiger  and 
Schmidt's  method  (see  p.  405). 

2.  Inorganic  Physiological  Constituents. 
Ammonia. 

Next  to  urea,  ammonia  is  the  most  important  of  the  nitrogenous 
end-products  of  protein  metabolism.  Ordinarily  about  4.6-5.6  per 
cent  of  the  total  nitrogen  of  the  urine  is  eliminated  as  ammonia 
and  on  the  average  this  would  be  about  0.7  gram  per  day.  Under 
normal  conditions  the  ammonia  is  present  in  the  urine  in  the  form  of 
the  chloride,  phosphate  or  sulphate.  This  is  due  to  the  fact  that 
combinations  of  this  sort  are  not  oxidized  in  the  organism  to  form 
urea,  but  are  excreted  as  such.  This  explains  the  increase  in  the 
output  of  ammonia  which  follows  the  administration  of  the  am- 
monium salts  of  the  mineral  acids  or  of  the  acids  themselves.  On 
the  other  hand  when  ammonium  acetate  and  many  other  ammonium 
salts  of  certain  organic  acids  are  administered  no  increase  in  the 

1  Magnesia  mixture  may  be  prepared  as  follows:  Dissolve  175  grams  of  MgS<  ' 
and  350  grams  of  NHiCl  in  1400  c.c.  of  distilled  water.  Add  700  grams  of 
concentrated  NH,OH,  mix  very  thoroughly  and  preserve  the  mixture  in  a  glass- 
stoppered  bottle. 

2  Ammoniacal  silver  solution  may  be  prepared  according  to  directions  given 
on  page  407. 


296  '     PHYSIOLOGICAL    CHEMISTRY. 

output  of  ammonia  occurs  since  the  salt  is  oxidized  and  its  nitrogen 
ultimately  appears  in  the  urine  as  urea. 

'  The  acids  formed  during  the  process  of  protein  destruction 
within  the  body  have  an  influence  upon  the  excretion  of  ammonia 
similar  to  that  exerted  by  acids  which  have  been  administered. 
Therefore  a  pathological  increase  in  the  output  of  ammonia  is  ob- 
served in  such  diseases  as  are  accompanied  by  an  increased  and  im- 
perfect protein  metabolism,  and  especially  in  diabetes,  in  which 
disease  diacetic  acid  and  /?-oxybutyric  acid  are  found  in  the  urine 
in  combination  with  the  ammonia. 

As  the  result  of  recent  experiments  Folin  claims  that  a  pro- 
nounced decrease  in  the  extent  of  protein  metabolism,  as  measured 
by  the  total  nitrogen  in  the  urine,  is  frequently  accompanied 
by  a  decreased  elimination  of  ammonia.  The  ammonia  elimination 
is  therefore  probably  determined  by  other  factors  than  the  total 
protein  catabolism  as  such.  Furthermore,  he  believes  that  a  de- 
cided decrease  in  the  total  nitrogen  excretion  is  always  accompanied 
by  a  relative  increase  in  the  ammonia-nitrogen,  provided  the  food  is 
of  a  character  yielding  an  alkaline  ash. 

The  quantitative  determination  of  ammonia  must  be  made  upon 
the  fresh  urine  since  upon  standing  the  normal  urine  will  undergo 
ammoniacal  fermentation  (see  page  257). 

Sulphates. 

Sulphur  in  combination  is  excreted  in  two  forms  in  the  urine ; 
first,  as  loosely  combined,  unoxidized  or  neutral  sulphur  and  second, 
as  oxidized  or  acid  sulphur.  The  loosely  combined  sulphur  is  ex- 
creted mainly  as  a  constituent  of  such  bodies  as  cystine,  cysteine, 
taurine,  hydrogen  sulphide,  ethyl  sulphide,  thiocyanates,  sul- 
phonic  acids,  oxyproteic  acid,  alloxpyroteic  acid  and  uroferric  acid. 
The  amount  of  loosely  combined  sulphur  eliminated  is  in  great 
measure  independent  of  the  extent  of  protein  decomposition  or  of 
the  total  sulphur  excretion.  In  this  characteristic  it  is  somewhat 
similar  to  the  excretion  of  creatinine.  The  oxidized  sulphur  is 
eliminated  in  the  form  of  sulphuric  acid,  principally  as  salts  of 
sodium,  potassium,  calcium  and  magnesium;  a  relatively  small 
amount  occurs  in  the  form  of  ethereal  sulphuric  acid,  i.  e.,  sulphuric 
acid  in  combination  with  such  aromatic  bodies  as  phenol,  indole, 
skatole,  cresol,  pyrocatechin  and  hydroquinone.  Sulphuric  acid  in 
combination  with  Na,  K,  Ca  or  Mg  is  sometimes  termed  inorganic 
or  preformed  sulphuric  acid  whereas  the  ethereal  sulphuric  acid  is 


URINE.  297 

sometimes  called  conjugate  sulphuric  acid.  The  greater  pan  of 
the  sulphur  is  eliminated  in  the  oxidized  form  but  the  absolute  per- 
centage of  sulphur  excreted  as  the  preformed,  ethereal  or  loosely 
combined  type  depends  upon  the  total  quantity  of  sulphur  present, 
i.  c,  there  is  no  definite  ratio  between  the  three  forms  of  sulphur 
which  will  apply  tinder  all  conditions.  The  preformed  sulphuric 
acid  may  he  precipitated  directly  from  acidified  urine  with  BaCL, 
whereas  the  ethereal  sulphuric  acid  must  undergo  a  preliminary 
boiling  in  the  presence  of  a  mineral  acid  before  it  can  he  so  precipi- 
tated. 

The  sulphuric  acid  excreted  by  the  urine  arises  principally  from 
the  oxidation  of  protein  material  within  the  body;  a  relatively  small 
amount  is  due  to  ingested  sulphates.  Under  normal  condition- 
about  2.5  grams  of  sulphuric  acid  is  eliminated  daily.  Since  the 
sulphuric  acid  content  of  the  urine  has,  for  the  most  part,  a  protein 
origin  and  since  one  of  the  most  important  constituents  of  the  pro- 
tein molecule  is  nitrogen,  it  would  be  reasonable  to  suppose  that  a 
fairly  definite  ratio  might  exist  betwen  the  excretion  of  these  two 
elements.  However,  when  we  appreciate  that  the  percentage  con- 
tent of  X  and  S  present  in  different  proteins  is  subject  to  rather 
wide  variations,  the  fixing  of  a  ratio  which  will  express  the  exact 
relation  existing  between  these  two  substances,  as  they  appear  in  the 
urine  as  end-products  of  protein  metabolism,  is  practically  im- 
possible. It  has  been  suggested  that  the  ratio  5:1  expresses  this 
relation  in  a  general  way. 

Pathologically,  the  excretion  of  sulphuric  acid  by  the  urine  is  in- 
creased in  acute  fevers  and  in  all  other  diseases  marked  by  a  stimu- 
lated metabolism,  whereas  a  decrease  in  the  sulphuric  acid  excretion 
is  observed  in  those  diseases  which  are  accompanied  by  a  loss  of 
appetite  and  a  diminished  metabolic  activity. 

Experiments. 

1.  Detection  of  Inorganic  Sulphuric  Acid. — Place  about  10  c.c. 
of  urine  in  a  test-tnbe.  acidify  with  acetic  acid  and  add  some  barium 
chloride  solution.      A  white  precipitate  of  barium  sulphate  forms. 

2.  Detection  of  Ethereal  Sulphuric  Acid. —  Filter  ^\'(  the 
barium  sulphate  precipitate  formed  in  the  above  experiment,  add 
1  c.c.  of  hydrochloric  acid  and  a  little  barium  chloride  solution  to 
the  filtrate  and  heat  the  mixture  to  boiling  for  [-2  minutes.  Note 
the  appearance  of  a  turbidity  due  to  the  presence  of  sulphuric  acid 


298 


PHYSIOLOGICAL    CHEMISTRY. 


Fig.  95. 


which  has  been  separated  from  the  ethereal  sulphates  and  has  com- 
bined with  the  barium  of  the  BaCl2  to  form  BaS04. 

3.  Detection  of  Loosely  Combined  or  Neutral  Sulphur. — 
Place  about  10  c.c.  of  urine  in  a  test-tube,  introduce  a  small  piece  of 
zinc,  add  sufficient  hydrochloric  acid  to  cause  a  gentle  evolution  of 
hydrogen  and  over  the  mouth  of  the  tube  place  a  filter  paper  satu- 
rated with  plumbic  acetate  solution.  In  a  short  time  the  portion  of 
the  paper  in  contact  with  the  vapors  within  the  test-tube  becomes 
blackened  due  to  the  formation  of  lead  sulphide.  The  nascent 
hydrogen  has  reacted  with  the  loosely  combined  or  neutral  sulphur 

to  form  hydrogen  sulphide  and  this 
gas  coming  in  contact  with  the  plum- 
bic acetate  paper  has  caused  the 
production  of  the  black  lead  sulphide. 
Sulphur  in  the  form  of  inorganic 
or  ethereal  sulphuric  acid  does  not 
respond  to  this  test. 

4.  Calcium  Sulphate  Crystals. — 
Place  10  c.c.  of  urine  in  a  test- 
tube,  add  10  drops  of  calcium  chlo- 
ride solution  and  allow  the  tube  to 
stand  until  crystals  form.  Examine 
the  calcium  sulphate  crystals,  under  the  microscope  and  compare 
them  with  those  shown  in  Fig.  95,  above. 


Calcium    Sulphate. 
and  Weil.) 


(Hensel 


Chlorides. 

Next  to  urea,  the  chlorides  constitute  the  chief  solid  constituent 
of  the  urine.  The  principal  chlorides  found  in  the  urine  are  those 
of  sodium,  potassium,  ammonium  and  magnesium,  with  sodium 
chloride  predominating:  The  excretion  of  chlorides  is  dependent, 
in  great  part,  upon  the  nature  of  the  diet,  but  on  the  average  the 
daily  output  is  about  10-15  grams,  expressed  as  sodium  chloride. 
Copious  water-drinking  increases  the  output  of  chlorides  consider- 
ably. Because  of  their  solubility,  chlorides  are  never  found  in  the 
urinary  sediment. 

Since  the  amount  of  chlorides  excreted  in  the  urine  is  due  pri- 
marily to  the  chloride  content  of  the  food  ingested,  it  follows  that 
a  decrease  in  the  amount  of  ingested  chloride  will  likewise  cause  a 
decrease  in  the  chloride' content  of  the  urine.  In  cases  of  actual 
fasting  the  chloride  content  of  the  urine  may  be  decreased  to  a 
slight    trace  which  is  derived  from    the    body  fluids  and  tissues. 


URINK.  299 

Under  these  conditions,  however,  an  examination  of  the-  blood  of 
the  fasting  subject  will  show  the  percentage  of  chlorides  in  this 
fluid  to  be  approximately  normal.  This  forms  a  very  striking  ex- 
ample of  the  care  nature  takes  to  maintain  the  normal  composition 
of  the  blood.  There  is  a  limit  to  the  power  of  the  body  to  main- 
tain this  equilibrium,  however,  and  if  the  fasting  organism  be  sub- 
jected to  the  influence  of  diuretics  for  a  time,  a  point  is  reached 
where  the  composition  of  the  blood  can  no  longer  be  maintained  and 
a  gradual  decrease  in  its  chloride  content  occurs  which  finally  re- 
sults in  death.  Death  is  supposed  to  result  not  so  much  because  of 
a  lack  of  chlorine  as  from  a  deficiency  of  sodium.  This  is  shown 
from  the  fact  that  potassium  chloride,  for  instance,  cannot  replace 
the  sodium  chloride  of  the  blood  when  the  latter  is  decreased  in  the 
manner  above  stated.  When  this  substitution  is  attempted  the 
potassium  salt  is  excreted  at  once  in  the  urine,  and  death  follows  as 
above  indicated. 

Pathologically,  the  excretion  of  chlorides  may  be  decreased  in 
some  fevers,  chronic  nephritis,  croupous  pneumonia,  diarrhoea,  cer- 
tain stomach  disorders  and  in  acute  articular  rheumatism. 

Experiment. 

Detection  of  Chlorides  in  Urine.— Place  about  5  c.c.  of  urine  in 
a  test-tube,  render  it  acid  with  nitric  acid  and  add  a  few  drops  of  a 
solution  of  argentic  nitrate.  A  white  precipitate,  due  to  the  forma- 
tion of  argentic  chloride,  is  produced.  This  precipitate  is  soluble 
in  ammonium  hydroxide. 

Phosphates. 

Phosphoric  acid  exists  in  the  urine  in  two  general  forms :  First, 
that  in  combination  with  the  alkali  metals,  sodium  and  potassium, 
and  the  radical  ammonium;  second,  that  in  combination  with  the 
alkaline  .earths,  calcium  and  magnesium.  Phosphates  formed 
through  a  union  of  phosphoric  acid  with  the  alkali  metals  are  termed 
alkaline  phosphates,  or  phosphates  of  the  alkali  metals,  whereas 
phosphates,  formed  through  a  union  of  phosphoric  acid  with  the 
alkaline  earths  are  termed  earthy  phosphates,  or  phosphates  of  the 
alkaline  earths. 

Three  series  of  salts  are  formed  by  phosphoric  acid :  Normal , 
M3P04,]  mono-hydrogen,  M2HP04,  and  di-hydrogen,  MH2P04. 
The  di-hydrogen  salts  are  acid  in  reaction  and  it  was  generally  be- 
lieved that  about  60  per  cent  of  the  total  phosphate  content  of  the 

1  M  may  be  occupied  by  any  of  the  alkali  metals  or  alkaline  earths. 


300  PHYSIOLOGICAL    CHEMISTRY. 

urine  was  in  the  form  of  this  type  of  salt,  and  that  the  acidity  of 
the  urine  was  due  in  great  part  to  the  presence  of  sodium  di-hydro-' 
gen  phosphate.  Recently,  however,  it  has  been  quite  clearly  shown 
that  the  normal  acidity  of  the  urine  is  not  due  to  the  presence  of  this 
salt  but  is  due,  at  least  in  part,  to  the  presence  of  various  acidic 
radicals.  In  this  connection  Folin  believes  that  the  phosphates  in 
clear  acid  urine  are  all  of  the  mono-hydrogen  type,  and  that  the 
acidity  of  the  urines  of  this  character  is  generally  greater  than 
the  combined  acidity  of  all  the  phosphates  present ;  the  excess  in 
the  acidity  above  that  due  to  phosphates  he  believes  to  be  due  to 
free  organic  acids.  The  observation  has  recently  been  made  that 
urine  may  be  separated  into  two  portions,  one  part  consisting  al- 
most entirely  of  inorganic  matter  including  practically  all  of  the 
phosphates  and  having  an  alkaline  reaction,  the  other  containing 
practically  all  of  the  organic  substances  and  no  phosphates  and  hav- 
ing an  acid  reaction. 

In  bones  the  phosphates  occur  principally  in  the  form  of  the  nor- 
mal salts  of  calcium  and  magnesium.  The  mono-hydrogen  salts  as 
a  class  are  alkaline  in  reaction  to  litmus,  and  it  is  to  the  presence  of 
di-sodium  hydrogen  phosphate,  Na2HP04,  that  the  greater  part  of 
the  alkalinity  of  the  saliva  is  due. 

The  excretion  of  phosphoric  acid  is  extremely  variable  but  on 
the  average  the  total  output  for  24  hours  is  about  2.5  grams,  ex- 
pressed as  P205.  Ordinarily  the  total  output  is  distributed  be- 
tween alkaline  phosphates  and  earthy  phosphates  approximately  in 
the  ratio  2:1.  The  greater  part  of  this  phosphoric  acid  arises  from 
the  ingested  food,  either  from  the  preformed  phosphates  or  more 
especially  from  the  phosphorus  in  organic  combination  such  as  we 
find  it  in  phosphoproteins,  nude 0 proteins  and  lecithins;  the  phos- 
phorus-containing tissues  of  the  body  also  contribute  to  the  total 
output  of  this  element.  Alkaline  phosphates  ingested  with  the  food 
have  a  tendency  to  increase  the  phosphoric  acid  content  of  the 
urine  to  a  greater  extent  than  the  earthy  phosphates  so  ingested. 
This  is  due,  in  a  measure,  to  the  fact  that  a  portion  of  the  earthy 
phosphates,  under  certain  conditions,  may  be  precipitated  in  the 
intestine  and  excreted  in  the  feces ;  this  is  especially  to  be  noted 
in  the  case  of  herbivorous  animals.  Since  the  extent  to  which  the 
phosphates  are  absorbed  in  the  intestine  depends  upon  the  form 
in  which  they  are  present  in  the  food,  under  ordinary  conditions, 
there  can  be  no  absolute  relationship  between  the  urinary  output 
of   nitrogen   and   phosphorus.     If   the   diet    is    constant   however, 


I 'KIN  K. 


30I 


from  day  to  day,  thus  allowing  of  the  preparation  of  both  a  nitrogen 
and  a  phosphorus  balance,3  a  definite  ratio  may  be  established.  In 
experiments  upon  dogs,  which  were  fed  an  exclusive  meat  diet,  the 
ratio  of  nitrogen  to  phosphorus,  in  the  urine  and  feces,  was  found 
to  be  8.1  :  1. 

Pathologically  the  excretion  of  phosphoric  acid  is  increased  in 
such  diseases  of  the  hones  as  diffuse  periostosis,  osteomalacia  and 
rickets;  according  to  some  investigators,  in  the  early  stages  of  pul- 
monary tuberculosis;  in  acute  yellow  atrophy  of  the  liver  :  in  diseases 
which  are  accompanied  by  an  extensive  decomposition  of  nervous 
tissue  and  after  sleep  induced  by  potassium  bromide  or  choral  hy 
drate  (Mendel).  It  is  also  increased  after  copious  water-drinking. 
A  decrease  in  the  excretion  of  phosphates  is  at  times  noted  in  febrile 
affections,  such  as  the  acute  infectious  diseases;  in  pregnancy,  in  the 
period  during  which  the  fcetal  hones  are  forming,  and  in  diseases 
of  the  kidneys,  because  of  non-elimination. 

Experiments. 

1.   Formation  of  "Triple  Phosphate." — Place  some  urine  in  a 
beaker,  render  it  alkaline  with  ammonium  hydroxide,  add  a  small 

Fig.  96. 


+ 


\ 


"  Tkiple  Phosphate."     |  Ogden.  I 


amount  of  magnesium  sulphate  solution  and  allow  the  beaker  to 
stand  in  a  cool  place  over  night.  Crystals  of  ammonium  magnesium 
phosphate,  "triple  phosphate,"  form  under  these  conditions.      Ex- 

1  In  metabolism  experiments,  a  statement  showing  the  relation  existing  between 
the  nitrogen  content  of  the  food  on  the  one  hand  and  that  of  the  mine  and  feces 
on  the  other,  for  a  definite  period,  is  termed  a  nitrogen   balance  or  a  "balance 

of  the  income  and  outgo  of  nitrogen." 


302  PHYSIOLOGICAL    CHEMISTRY. 

amine  the  crystalline  sediment  under  the  microscope  and  compare 
the  forms  of  the  crystals  with  those  shown  in  Fig.  96,  page  301. 

2.  "  Triple  Phosphate  "  Crystals  in  Ammoniacal  Fermenta- 
tion.— Stand  some  urine  aside  in  a  beaker  for  several  days.  Am- 
moniacal fermentation  will  develop  and  "  triple  phosphate  "  crystals 
will  form.  Examine  the  sediment  under  the  microscope  and  com- 
pare the  crystals  with  those  shown  in  Fig.  96,  p.  301. 

3.  Detection  of  Earthy  Phosphates. — Place  10  c.c.  of  urine 
in  a  test-tube  and  render  it  alkaline  with  ammonium  hydroxide. 
Warm  the  mixture  and  note  the  separation  of  a  precipitate  of  earthy 
phosphates. 

4.  Detection  of  Alkaline  Phosphates. — Filter  off  the  earthy 
phosphates  as  formed  in  the  last  experiment,  and  add  a  small  amount 
of  magnesia  mixture  (see  page  295)  to  the  filtrate.  Now  warm 
the  mixture  and  observe  the  formation  of  a  white  precipitate  due 
to  the  presence  of  alkaline  phosphates.  Note  the  difference  in  the 
size  of  the  precipitates  of  the  two  forms  of  phosphates  from  this 
same  volume  of  urine.  Which  form  of  phosphates  were  present 
in  the  larger  amount,  earthy  or  alkaline ? 

5.  Influence  upon  Fehling's  Solution. — Place  2  c.c.  of  Fell- 
ling's  solution  in  a  test-tube,  dilute  it  with  4  volumes  of  water  and 
heat  to  boiling.  Add  a  solution  of  sodium  dihydrogen  phosphate, 
NaH2P04,  a  small  amount  at  a  time,  and  heat  after  each  addition. 
What  do  you  observe?  What  does  this  observation  force  you  to' 
conclude  regarding  the  interference  of  phosphates  in  the  testing  of 
diabetic  urine  by  means  of  Fehling's  test? 

Sodium  and  Potassium. 

The  elements  sodium  and  potassium  are  always  present  in  the 
urine.  Usually  they  are  combined  with  such  acidic  radicals  as 
CI,  C03,  S04  and  P04.  The  amount  of  potassium,  expressed  as 
K20,  excreted  in  24  hours  by  an  adult,  subsisting  upon  a  mixed 
diet,  is  on  the  average  2-3  grams,  whereas  the  amount  of  sodium, 
expressed  as  Na20,  under  the  same  conditions,  is  ordinarily  4-6 
grams.  The  ratio  of  K  to  Na  is  generally  about  3:5.  The  ab- 
solute quantity  of  these  elements  excreted,  depends,  of  course,  in 
large  measure,  upon  the  nature  of  the  diet.  Because  of  the  non- 
ingestion  of  NaCl  and  the  accompanying  destruction  of  potassium- 
containing  body  tissues,  the  urine  during  fasting  contains  more 
potassium  salts  than  sodium  salts. 

Pathologically  the  output  of  potassium,  in  its  relation  to  sodium, 


URINE.  303 

may  be  increased  during  fever;  following  the  crisis,  however,  the 
output  of  this  element  may  be  decreased.  It  may  also  he  increased 
in  conditions  associated    with   acid   intoxication. 

Calcium  and  Magnesium. 

The  greater  part  of  the  calcium  and  magnesium  excreted  in 
the  urine  is  in  the  form  of  phosphates.  The  daily  output,  which 
depends  principally  upon  the  nature  of  the  diet,  aggregates  ori 
the  average  about  1  gram  and  is  made  up  of  the  phosphate-  of  cal- 
cium and  magnesium  in  the  proportion  1  :  2.  The  percentage  of 
calcium  salts  present  in  the  urine  at  any  one  time  forms  no  depend 
able  index  as  to  the  absorption  of  this  class  of  salts,  since  they  arc- 
again  excreted  into  the  intestine  after  absorption.  It  is  therefore 
impossible  to  draw  any  satisfactory  conclusions  regarding  the  ex- 
cretion of  the  alkaline  earths  unless  we  obtain  accurate  analytical 
data  from  both  the  feces  and  the  urine. 

Very  little  is  known  positively  regarding  the  actual  course  of  the 
excretion  of  the  alkaline  earths  under  pathological  conditions  except 
that  an  excess  of  calcium  is  found  in  acid  intoxication  and  some 
diseases  of  the  bones. 

Carbonates. 

Carbonates  generally  occur  in  small  amount  in  the  urine  of  man 
and  carnivora  under  normal  conditions,  whereas  much  larger  quan- 
tities are  ordinarily  present  in  the  urine  of  herbivora.  The  alkaline 
reaction  of  the  urine  of  herbivora  is  dependable  in  great  measure 
upon  the  presence  of  carbonates.  In  general  a  urine  containing 
carbonates  in  appreciable  amount  is  turbid  when  passed  or  becomes 
so  shortly  after.  These  bodies  ordinarily  occur  as  alkali  or  alkaline 
earth  compounds  and  the  turbid  character  of  urine  containing  them 
is  usually  due  principally  to  the  latter  class  of  substances.  The 
carbonates  of  the  alkaline  earths  are  often  found  in  amorphous 
urinary  sediments. 

Iron. 

Iron  is  present  in  small  amount  in  normal  urine.  It  probably 
occurs  partly  in  inorganic  and  partly  in  organic  combination.  The 
iron  contained  in  urinary  pigments  or  chromogens  is  in  organic  com- 
bination. According  to  different  investigators  the  iron  content  of 
normal  urine  will  probably  not  average  more  than  0.001  gram 
per  day. 


304  physiological  chemistry. 

Experiment. 

Detection  of  Iron  in  Urine. — Evaporate  a  convenient  volume 
( 10-15  c.c.)  of  urine  to  dryness.  Incinerate  and  dissolve  the  residue 
in  a  few  drops  of  iron-free  hydrochloric  acid  and  dilute  the  acid 
solution  with  5  c.c.  of  water.  Divide  the  acid  solution  into  two 
parts  and  make  the  following  tests:  (a)  To  the  first  part  add  a 
solution 'of  ammonium  thiocyanate;  a  red  color  indicates  the  pres- 
ence of  iron,  (b)  To  the  second  part  of  the  solution  add  a  little 
potassium  ferrOcyanide  solution ;  a  precipitate  of  Prussian  blue 
forms  upon  standing. 

Fluorides,  Nitrates,  Silicates  and  Hydrogen  Peroxide. 

These  substance  are  all  found  in  traces  in  human  urine  under 
normal  conditions.  Nitrates  are  undoubtedly  introduced  into  the 
organism  in  the  water  and  ingested  food.  The  average  excre- 
tion of  nitrates  is  about  0.5  gram  per  day,  the  output  being  the 
larg'est  upon  a  vegetable  diet  and  smallest  upon  a  meat  diet.  Ni- 
trites are  found  only  in  urine  which  is  undergoing  decomposition 
and  are  formed  from  the  nitrates  in  the  course  of  ammoniacal 
fermentation.  Hydrogen  peroxide  has  been  detected  in  the  urine, 
but  its  presence  is  believed  to  possess  no  pathological  importance. 


CHATTER    XIX. 

URINE:    PATHOLOGICAL    CONSTITUENTS.' 

Dextrose. 

Serum  albumin. 
Serum  globulin. 


Proteins 


Proteoses 


Blood 


Bile 


Deutero-proteose. 
Hetero-proteose. 
"  Bence- Jones'  protein. 
Peptone. 
Nucleoprotein. 
Fibrin. 

Oxyhemoglobin. 
Form  elements. 
Pigment, 
f  Pigments. 
I  Acids. 


Acetone. 

Diacetic  acid. 

/3-Oxy butyric  acid. 

Conjugate  glycuronates. 

Pentoses. 

Fat. 

Ha?matoporphy  rin . 

Lactose. 

Galactose. 

Lasvulose. 

Inosite. 

Laiose. 

Melanin. 

Urorosein. 

Unknown  substances. 


DEXTROSE. 

Traces  of  this  sugar  occur  in  normal  urine,  but  the  amount  is  not 
sufficient  to  be  readily  detected  by  the  ordinary  simple  qualitative 
1  See  note  at  the  bottom  of  page  264. 
21  305 


306  PHYSIOLOGICAL    CHEMISTRY. 

tests.  There  are  two  distinct  types  of  pathological  glycosuria,  i.  e., 
transitory  glycosuria  and  persistent  glycosuria.  The  transitory 
type  may  follow  the  ingestion  of  an  excess  of  sugar,  causing 
the  assimilation  limit  to  be  exceeded,  or  it  may  accompany 
any  one  of  several  disorders  which  cause  an  impairment  of  the 
power  of  assimilating  sugar.  In  the  persistent  type  large  amounts 
of  sugar  are  excreted  daily  in  the  urine  for  long-  periods  of  time. 
Under  such  circumstances  a  condition  known  as  diabetes  mellitus 
exists.  Ordinarily,  diabetic  urine  which  contains  a  high  percentage 
of  sugar  possesses  a  faint  yellow  color,  a  high  specific  gravity  and 
a  volume  which  is  above  normal. 

Experiments. 

i  .  Phenylhydrazine  Reaction. — Test  the  urine  according  to  one 
of  the  following  methods :  (a)  To  a  small  amount  of  phenylhy- 
drazine mixture,  furnished  by  the  instructor,1  add  5  c.c.  of  the  urine, 
shake  well  and  heat  on  a  boiling  water-bath  for  one-half  to  three- 
quarters  of  an  hour.  Allow  the  tube  to  cool  slowly  and  examine 
the  crystals  microscopically  (Plate  III.,  opposite  page  24).  If  the 
solution  has  become  too  concentrated  in  the  boiling  process  it  will 
be  light-red  in  color  and  no  crystals  will  separate  until  it  is  diluted 
with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  cer- 
tain sugars  under  these  conditions,  in  general  each  individual  sugar 
giving  rise  to  an  osazone  of  a  definite  crystalline  form  which  is 
typical  for  that  sugar.  It  is  important  to  remember  in  this  con- 
nection that,  of  the  simple  sugars  of  interest  in  physiological  chem- 
istry, dextrose  and  lsevulose  yield  the  same  osazone,  with  phenylhy- 
drazine. Each  osazone  has  a  definite  melting-point,  and  as  a  fur- 
thur  and  more  accurate  means  of  identification  it  may  be  recrys- 
tallized  and  identified  by  the  determination  of  its  melting-point  and 
nitrogen  content.  The  reaction  taking  place  in  the  formation  of 
phenyldextrosazone  is  as  follows : 

C6H1206  +  2(H2N-NH-C6H5)  = 

Dextrose.  Phenylhydrazine. 

C6H10O4(NNHC6H5)2  +  2H20  +  H2. 

Phenyldextrosazone. 

(b)   Place  5  c.c.  of  the  urine  in  a  test-tube,  add  1  c.c.  of  phen- 

1  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazine-hydro- 
chloride  and  two  parts  of  sodium  acetate,  by  weight.  These  are  thoroughly 
mixed  in  a  mortar. 


URINE.  307 

ylhydrazine-acetate  solution  furnished  by  the  instructor,1  and  heat 
on  a  boiling  water-bath  for  one-half  to  three-quarters  of  an  hour. 
Allow  the  liquid  to  cool  slozvly  and  examine  the  crystals  microscop- 
ically (Plate  III.,  opposite  p.  24). 

The  phenylhydrazine  test  has  been  so  modified  by  Cipollina  as  to 
be  of  use  as  a  rapid  clinical  test.  The  directions  for  this  test  are 
given  in  the  next  experiment. 

2.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  urine,  5  drops 
of  phenylhydrazine  (the  base)  and  one-half  c.c.  of  glacial  acetic 
acid  in  a  test-tube.  Heat  the  mixture  for  about  one  minute  over 
a  low  flame,  shaking  the  tube  continually  to  prevent  loss  of  fluid 
by  bumping.  Add  4-5  drops  of  potassium  hydroxide  or  sodium 
hydroxide  (sp.  gr.  1.16),  being  certain  that  the  fluid  in  the  test- 
tube  remains  acid ;  heat  the  mixture  again  for  a  moment  and  then 
cool  the  contents  of  the  tube.  Ordinarily  the  crystals  form  at 
once,  especially  if  the  urine  possesses  a  low  specific  gravity.  If  they 
do  not  appear  immediately  allow  the  tube  to  stand  at  least  20  min- 
utes before  deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  shown  in  Plate  III.,  opposite  page  24. 

3.  Reduction  Tests. — To  their  aldehyde  or  ketone  structure 
many  sugars  owe  the  property  of  readily  reducing  the  alkaline 
solutions  of  the  oxides  of  metals  like  copper,  bismuth  and  mercury; 
they  also  possess  the  property  of  reducing  ammoniacal  silver  solu- 
tions with  the  separation  of  metallic  silver.  Upon  this  property 
of  reduction  the  most  widely  used  tests  for  sugars  are  based. 
When  whitish-blue  cupric  hydroxide  in  suspension  in  an  alkaline 
liquid  is  heated  it  is  converted  into  insoluble  black  cupric  oxide,  but 
if  a  reducing  agent  like  certain  sugars  be  present  the  cupric  hy- 
droxide is  reduced  to  insoluble  yellow  cuprous  hydroxide,  which 
in  turn  on  further  heating  may  be  converted  into  brownish-red 
or  red  cuprous  oxide.     These  changes  are  indicated  as  follows : 

OH 

/ 
Cu  ^—     Cu  ==  0  +  H20. 

\  Cupric  oxide 

\  (black  1. 

OH 

Cupric  hydroxide 
(whitish-blue). 

1  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case,  of 
glacial  acetic  acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the 
base). 


3o8 


PHYSIOLOGICAL    CHEMISTRY. 

OH 


Cu 


/ 

i 

\ 


Cu 


\ 


OH 
OH 

OH 


2Cu  -  OH  +  H20  +  0. 

Cuprous  hydroxide 
(yellow). 


Cu 


Cu-OH 
Cu-OH 


Cu 


\ 

( 

/ 


0  +  H20. 


Cuprous  hydroxide  Cuprous  oxide 

(yellow).  (brownish-red). 

The  chemical  equations  here  discussed  are  exemplified  in  Trom- 
mer's and  Fehling's  tests. 

(a)  Trommer's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  one-, 
half  its  volume  of  KOH  or  NaOH.  Mix  thoroughly  and  add,  drop 
by  drop,  agitating  after  the  addition  of  each  drop,  a  very  dilute 
solution  of  cupric  sulphate.  Continue  the  addition  until  there  is 
a  slight  permanent  precipitate  of  cupric  hydroxide  and  in  con- 
sequence the  solution  is  slightly  turbid.  Heat,  and  the  cupric  hy- 
droxide is  reduced  to  yellow  cuprous  hydroxide  or  to  brownish-red 
cuprous  oxide.  If  the  solution  of  cupric  sulphate  used  is  too  strong, 
a  small  brownish-red  precipitate  produced  in  the  presence  of  a 
low  percentage  of  dextrose  may  be  entirely  masked.  On  the  other 
hand,  if  too  little  cupric  sulphate  is  used  a  light-colored  precipitate 
formed  by  uric  acid  and  purine  bases  may  obscure  the  brownish- 
red  precipitate  of  cuprous  oxide.  The  action  of  KOH  or  NaOH 
in  the  presence  of  an  excess  of  sugar  and  insufficient  copper  will 
produce  a  brownish  color.  Phosphates  of  the  alkaline  earths  may 
also  be  precipitated  in  the  alkaline  solution  and  be  mistaken  for 
cuprous  hydroxide.     Trommer's  test  is  not  very  satisfactory. 

(b)  Fehling's  Test. — To  about  1  c.c.  of  Fehling's  solution1   in 

1  Fehling's  solution  is  composed  of  two  definite  solutions — a  cupric  sulphate 
solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows : 

Cupric  sulphate  solution  =  34.65  grams  of  cupric  sulphate  dissolved  in  water 
and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  125  grams  of  potassium  hydroxide  and  173 
grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 


URINE.  309 

a  test-tube  add  about  4  c.c.  of  water,  and  boil.  This  is  done  to 
determine  whether  the  solution  will  of  itself  cause  the  formation 
of  a  precipitate  of  brownish-red  cuprous  oxide.  If  such  a  pre- 
cipitate forms,  the  behling's  solution  must  not  be  used.  Add 
urine  to  the  warm  Fehling's  solution,  a  few  drops  at  a  tunc,  and 
heat  the  mixture  after  each  addition.  'The  production  of  yellow 
cuprous  hydroxide  or  brownish-red  cuprous  oxide  indicates  that 
reduction  has  taken  place.  The  yellow  precipitate  is  more  likely 
to  occur  if  the  urine  is  added  rapidly  and  in  large  amount,  whereas 
with  a  less  rapid  addition  of  smaller  amounts  of  urine  the  brownish- 
red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even 
this  test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the 
urine.  Such  bodies  as  conjugate  giycuronates,  uric  acid,  nucleo- 
protein  and  homogentisic  acid,  when  present  in  sufficient  amount, 
may  produce  a  result  similar  to  that  produced  by  sugar.  Phosphates 
of  the  alkaline  earths  may  be  precipitated  by  the  alkali  of  the  Feh- 
ling's  solution  and  in  appearance  may  be  mistaken  for  the  cuprous 
diydroxide.  Cupric  hydroxide  may  also  be  reduced  to  cuprous 
oxide  and  this  in  turn  be  dissolved  by  creatinine^  a  normal  urinary 
constituent.  This  will  give  the  urine  under  examination  a  greenish 
tinge  and  may  obscure  the  sugar  reaction  even  when  a  considerable 
amount  of  sugar  is  present. 

(c)  Benedict's  Modifications  of  Fehling's  Test. — First  Modifi- 
cation.— To  2  c.c.  of  Benedict's  solution1  in  a  test-tube  add  6  c.c. 
of  distilled  water  and  7-9  drops  (not  more)  of  the  urine  under 
examination.  Boil  the  mixture  vigorously  for  about  15-30  sec- 
onds and  permit  it  to  cool  to  room  temperature  spontaneously.  ( If 
desired  this  process  may  be  repeated,  although  it  is  ordinarily  un- 
necessary.) If  sugar  is  present  in  the  solution  a  precipitate  will 
form  which  is  often  bluish-green  or  green  at  first,  especially  if  the 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and 
mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to  prevent  de- 
terioration. 

1  Benedict's  modified  Fehling  solution  consists  of  two  definite  solution? — a 
cupric  sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be  pre- 
pared as  follows : 

Cupric  sulphate  solution  =  34.65  grams  of  cupric  sulphate  dissolved  in  water 
and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  100  grams  of  anhydrous  sodium  carbonate  and 
173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppefed  bottles 
and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to  prevent 
deterioration. 


3IO  PHYSIOLOGICAL    CHEMISTRY. 

percentage  of  sugar  is  low,  and  which  usually  becomes  yellozvish 
upon  standing.  If  the  sugar  present  exceeds  0.06  per  cent  this 
precipitate  generally  forms  at  or  below  the  boiling-point,  whereas 
if  less  than  0.06  per  cent  of  sugar  is  present  the  precipitate  forms 
more  slowly  and  generally  only  after  the  solution  has  cooled.  The 
greenish  precipitate  obtained  with  urines  containing  small  amounts 
of  sugar  may  be  a  compound  of  copper  with  the  sugar  or  a  com- 
pound of  some  constituent  of  the  urine  with  reduced  copper  oxide 
instead  of  being  a  precipitate  of  cuprous  hydroxide  or  oxide  as  is 
the  case  when  the  original  Fehfing  solution  is  reduced. 

Benedict  claims  that,  whereas  the  original  Fehling  test  will  not 
serve  to  detect  sugar  when  present  in  a  concentration  of  less  than 
0.1  per  cent  that  the  above  modification  will  serve  to  detect  sugar 
when  present  in  as  small  quantity  as  0.015-0.02  per  cent.  The 
modified  solution  used  in  the  above  test  differs  from  the  original  in 
that  100  grams  of  sodium  carbonate  is  substituted  for  the  125 
grams  of  potassium  hydroxide  ordinarily  used,  thus  forming  a 
Fehling  solution  which  is  considerably  less  alkaline  than  the  orig- 
inal. This  alteration  in  the  composition  of  the  Fehling  solution 
is  of  advantage  in  the  detection  of  sugar  in  the  urine  inasmuch  as 
the  strong  alkalinity  of  the  ordinary  Fehling  solution  has  a  tendency, 
when  the  reagent  is  boiled  with  a  urine  containing  a  small  amount 
of  dextrose,  to  decompose  sufficient  of  the  sugar  to  render  the  de- 
tection of  the  remaining  portion  exceedingly  difficult  by  the  usual 
technique.  Benedict  claims  that  for  this  reason  the  use  of  his  mod- 
ified solution  permits  the  detection  of  smaller  amounts  of  sugar  than 
Joes  the  use  of  the  ordinary  Fehling  solution.  Benedict  has  fur- 
ther modified  his  solution  for  use  in  the  quantitative  determination 
of  sugar  (see  page  368). 

Second  Modification.1 — Very  recently  Benedict  has  further  modi- 
fied his  solution  and  has  succeeded  in  obtaining  one  which  does  not 
deteriorate  upon  long  standing.2     The  following  is  the  procedure 

1  Private  communication  from  Dr.  S.  R.  Benedict. 

2  Benedict's  new  solution  has  the  following  composition  : 

Cupric   sulphate    17.3  gm. 

Sodium  citrate I73-0  gm. 

Sodium    carbonate    (anhydrous)     100.0  gm. 

Distilled   water   to    1000.0   c.c. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600 
c.c.  of  water.  Pour  (through  a  folded  filter  if  necessary)  into  a  glass  graduate 
and  make  up  to  850  c.c.  Dissolve  the  cupric  sulphate  in  about  100  c.c.  of  water 
and  make  up  to  150  c.c.  Pour  the  carbonate-citrate  solution  into  a  large  beaker 
or  casserole  and  add  the  cupric  sulphate  solution  slowly,  with  constant  stirring. 
The  mixed  solution  is  ready  for  use,  and  does  not  deteriorate  upon  long  standing. 


URINE.  31  I 

"for  the  detection  of  dextrose  in  the  urine :  To  five  cubic  centimeters 
of  the  reagent  in  a  test-tube  add  eight  (not  more)  drops  of  the 
urine  to  be  examined.  The  fluid  is  then  boiled  vigorously  for  from 
one  to  two  minutes  and  then  allowed  to  cool  spontaneously.  In  the 
presence  of  dextrose  the  entire  body  of  the  solution  will  be  filled 
with  a  precipitate,  which  may  be  red,  yellow  or  green  in  color, 
depending  upon  the  amount  of  sugar  present.  [f  no  dextrose  is 
present,  the  solution  will  either  remain  perfectly  clear,  or  will  show 
a  very  faint  turbidity,  due  to  precipitated  urates.  Even  very  small 
quantities  of  dextrose  in  urine  (0.1  per  cent)  yield  precipitates  of 
surprising  bulk  with  this  reagent,  and  the  positive  reaction  for  dex- 
trose is  the  filling  of  the  entire  body  of  the  solution  with  a  precipi- 
tate, so  that  the  solution  becomes  opaque.  Since  amount  rather 
than  color  of  the  precipitate  is  made  the  basis  of  this  test,  it  may 
be  applied,  even  for  the  detection  of  small  quantities  of  dextrose, 
as  readily  in  artificial  light  as  in  daylight. 

(d)  Allen's  Modification  of  Fehling's  Test. — The  following  pro- 
cedure is  recommended :  "  From  7  to  8  c.c.  of  the  sample  of  urine 
to  be  tested  is  heated  to  boiling  in  a  test-tube,  and,  without  separat- 
ing any  precipitate  of  albumin  which  may  be  produced,  5  c.c.  of 
the  solution  of  cupric  sulphate  used  for  preparing  Fehling's  solution 
is  added.  This  produces  a  precipitate  containing  uric  acid,  xanthine, 
hypoxanthine,  phosphates,  etc.  To  render  the  precipitation  complete, 
however,  it  is  desirable  to  add  to  the  liquid,  when  partially  cooled. 
from  1  to  2  c.c.  of  a  saturated  solution  of  sodium  acetate  having 
a  feebly  acid  reaction  to  litmus.1  The  liquid  is  filtered  and  to  the 
filtrate,  which  will  have  a  bluish-green  color,  5  c.c.  of  the  alkaline 
tartrate  mixture  used  for  preparing  Fehling's  solution  is  added, 
and  the  liquid  boiled  for  15-20  seconds.  In  the  presence  of  mure 
than  0.25  per  cent  of  sugar,  separation  of  cuprous  oxide  occurs 
before  the  boiling-point  is  reached;  but  with  smaller  quantities 
precipitation  takes  place  during  the  cooling  of  the  solution,  which 
becomes  greenish,  opaque,  and  suddenly  deposits  cuprous  oxide  as 
a  fine  brownish-red  precipitate." 

(e)  Boettger's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  1  c.c 

1  Sufficient  acetic  acid  should  be  added  to  the  sodium  acetate  solution  to  render 
it  feebly  acid  to  litmus.  A  saturated  solution  of  sodium  acetate  keeps  well,  but 
weaker  solutions  are  apt  to  become  mould}-,  and  then  possess  the  power  of 
reducing  Fehling's  solution.  Hence  it  is  essential  in  all  cases  of  importance  to 
make  a  blank  test  by  mixing  equal  measures  of  cupric  sulphate  solution, 
alkaline  tartrate  solution  and  water,  adding  a  little  sodium  acetate  solution, 
and  heating  the  mixture  to  boiling. 


312  PHYSIOLOGICAL    CHEMISTRY. 

of  KOH  or  NaOH  and  a  very  small  amount  of  bismuth  subnitrate, 
and  boil.  The  solution  will  gradually  darken  and  finally  assume 
a  black  color  due  to  reduced  bismuth.  If  the  test  is  made  with 
urine  containing  albumin  this  must  be  removed,  by  boiling  and  fil- 
tering, before  applying  the  test,  since  with  albumin  a  similar  change 
of  color  is  produced   (bismuth  sulphide). 

(/)  Nylander's  Test  (Ahnen's  Test). — To  5  c.c.  of  urine  in  a 
test-tube  add  one-tenth  its  volume  of  Nylander's  reagent1  and  heat 
for  five  minutes  in  a  boiling  water-bath.2  The  mixture  will  darken 
if  reducing  sugar  is  present  and  upon  standing  for  a  few  moments 
a  black  color  will  appear.  This  color  is  clue  to  the  precipitation 
of  bismuth.  If  the  test  is  made  on  urine  containing  albumin  this 
must  be  removed,  by  boiling  and  filtering,  before  applying  the 
test  since  with  albumin  a  similar  change  of  color  is  produced. 
Dextrose  when  present  to  the  extent  of  0.08  per  cent  may  be  de- 
tected by  this  reaction.  It  is  claimed  by  Bechold  that  Nylander's 
and  Boettger's  tests  give  a  negative  reaction  with  solutions  con- 
taining sugar  when  mercuric  chloride  or  chloroform  is  present. 
Other  observers  have  failed  to  verify  the  inhibitory  action  of  the 
mercuric  chloride  and  have  shown  that  the  inhibitory  influence  of 
chloroform  may  be  overcome  by  raising  the  temperature  of  the 
urine  ,to  the  boiling-point  for  a  period  of  five  minutes  previous  to 
making  the  test. 

Urines  rich  in  indican,  uroerythrin  or  h(E  mat  0  porphyrin,  as  well 
as  urines  excreted  after  the  ingestion  of  large  amounts  of  certain 
medicinal  substances,  may  give  a  darkening  of  Nylander's  reagent 
similar  to  that  of  a  true  sugar  reaction. 

According  to  Rustin  and  Otto  the  addition  of  PtCl4  increases  the 
delicacy  of  Nylander's  reaction.  They  claim  that  this  procedure 
causes  the  sugar  to  be  converted  quantitatively.  No  quantitative 
method  has  yet  been  devised,  however,  based  upon  this  principle. 

A  positive  Nylander  or  Boettger  test  is  probably  due  to  the  fol- 
lowing reactions : 

(a)         Bi(OH)2N03  +  KOH  =  Bi(OH)3  +  KNO„. 
(6)  2Bi(OH)3  -  30  =  Bi2  +  3H20. 

4.  Fermentation  Test. — Rub  up  in  a  mortar  about  15  c.c.  of 

1  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  subnitrate 
and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium  hydroxide 
solution.    The  reagent  is  then  cooled  and  filtered. 

3  Hammarsten  suggests  that  the  solution  be  boiled  for  2-5  minutes  (accord- 
ing to  the  sugar  content)  over  a  free  flame  and  the  tube  then  permitted  to 
stand  five  minutes  before  drawing  conclusions. 


URINE.  J  '  3 

the  urine  with  a  small  piece  of  compressed  yeast.  Transfer  the 
mixture  to  a  saccharometer  (  Fig.  -'.  p.  3]  )  and  stand  it  aside  in 
a  warm  place  for  about  12  hours.  It  dextrose  is  present,  alco- 
holic fermentation  will  occur  and  carbon  dioxide  will  colled  as  a 

gas  in  the  upper  portion  of  the  tube.     On  the  completion  of   fer- 
mentation introduce,  by  means  of  a  bent  pipette,  a  little  ECOH    50 
lution  into  the  graduated  portion,  place  the  thumb  tightly  over  the 
opening  in  the  apparatus  and  invert  the  saccharometer.      Explain 
the  result. 

5.  Barfoed's  Test. — Place  about  5  c.c.  of  Barfoed's  solution1  in 
a  test-tube  and  heat  to  boiling.  Add  the  urine  under  examination 
slowly,  a  few  drops  at  a  time,  heating  after  each  addition.  Re- 
duction is  indicated  by  the  production  of  a  red  precipitate.  If  the 
precipitate  does  not  form  upon  continued  boiling  allow  the  tube  to 
stand  a  few  minutes  and  examine.  XaCl  interferes  with  this  test 
(Welker). 

Barfoed's  test  is  not  a  specific  test  for  dextrose  as  is  frequently 
stated,  but  simply  serves  to  detect  monosaccharides.  Disaccharides 
will  also  respond  to  the  test,  according  to  Hinkel  and  Sherman,  if 
the  solution  is  boiled  sufficiently  long  in  contact  with  the  reagent 
to  hydrolyze  the  disaccharide  through  the  action  of  the  acetic  acid 
present  in  the  Barfoed's  solution. 

6.  Polariscopic  Examination. — For  directions  as  to  the  use  of 
the  polariscope  see  page  32. 

PROTEINS. 

Normal  urine  contains  a  trace  of  protein  material  but  the  amount 
present  is  so  slight  as  to  escape  detection  by  any  of  the  simple  tests 
in  general  use  for  the  detection  of  protein  urinary  constituents. 
The  following  are  the  more  important  forms  of  protein  material 
which  have  been  detected  in  the  urine  under  pathological  conditions  : 

( 1 )  Serum  albumin. 

(2)  Serum  globulin. 

I  Deutero-proteose. 

(3)  Proteoses  -i  Hetero-proteose. 

I  "  Bence-Jones'  protein." 

(4)  Peptone. 

(5)  Nucleoprotein. 

(6)  Fibrin. 

(7)  Oxyhemoglobin. 

'Barfoed's  solution  is  prepared  as  follow-:  Dissolve  4.5  grams  of  neutral, 
crystallized  cupric  acetate  in  too  c.c.  of  water  and  add  0.12  c.c.  of  50  per  cent 
acetic  acid. 


314  PHYSIOLOGICAL    CHEMISTRY. 

ALBUMIN. 

Albuminuria  is  a  condition  in  which  serum  albumin  or  serum 
globulin  appears  in  the  urine.  There  are  two  distinct  forms  of 
albuminuria,  i.  e.,  renal  albuminuria  and  accidental  albuminuria. 
Sometimes  the  terms  "true"  albuminuria  and  "false"  albuminuria 
are  substituted  for  those  just  given.  In  the  renal  type  the  albumin 
is  excreted  by  the  kidneys.  This  is  the  more  serious  form  of  the 
malady  and  at  the  same  time  is  more  frequently  encountered  than 
the  accidental  type.  Among  the  causes  of  renal  albuminuria  are 
altered  blood  pressure  in  the  kidneys,  altered  kidney  structure,  or 
changes  in  the  composition  of  the  blood  entering  the  kidneys,  thus 
allowing  the  albumin  to  diffuse  more  readily.  In  the  accidental 
form  of  albuminuria  the  albumin  is  not  excreted  by  the  kidneys  as 
is  the  case  in  the  renal  form  of  the  disorder,  but  arises  from  the 
blood,  lymph  or  some  albumin-containing  exudate  coming  into  con- 
tact with  the  urine  at  some  point  below  the  kidneys. 

Experiments. 

i.  Heller's  Ring  Test. — Place  5  c.c.  of  concentrated  HN03  in  a 
test-tube,  incline  the  tube,  and,  by  means  of  a  pipette  allow  the 
urine  to  flow  slowly  down  the  side.  The  liquids  should  stratify 
with  the  formation  of  a  white  zone  of  precipitated  albumin  at  the 
point  of  juncture.  If  the  albumin  is  present  in  very  small  amount 
the  white  zone  may  not  form  until  the  tube  has  been  allowed  to 
stand  for  several  minutes.  If  the  urine  is  quite  concentrated  a 
white  zone,  due  to  uric  acid  or  urates,  will  form  upon  treatment  with 
nitric  acid  as  indicated.  This  ring  may  be  easily  differentiated 
from  the  albumin  ring  by  repeating  the  test  after  diluting  the  urine 
with  3  or  4  volumes  of  water,  whereupon,  the  ring,  if  due  to  uric 
acid  or  urates,  will  not  appear.  It  is  ordinarily  possible  to  differ- 
tiate  between  the  albumin  ring  and  the  uric  acid  ring  without 
diluting  the  urine,  since  the  ring,  when  due  to  uric  acid,  has  ordi- 
narily a  less  sharply  defined  upper  border,  is  generally  broader  than 
the  albumin  ring  and  frequently  is  situated  in  the  urine  above  the 
point  of  contact  with  the  nitric  acid.  Concentrated  urines  also  oc- 
casionally exhibit  the  formation,  at  the  point  of  contact,  of  a  crys- 
talline ring  with  very  sharply  defined  borders.  This  is  urea  nitrate 
and  is  easily  distinguished  from  the  "fluffy"  ring  of  albumin.  If 
there  is  any  difficulty  in  differentiation  a  simple  dilution  of  the  urine 
with  water,  as  above  described,  will  remove  the  difficulty.     Various 


URINE.  315 

colored  zones,  due  either  to  the  presence  of  indican,  bile  pigments  or 
to  the  oxidation  of  other  organic  urinary  constituents,  may  form  in 

this  test  under  certain  conditions.  These  colored  rings  should 
never  be  confounded  with  the  white  ring  which  alone  denotes  the 
presence  of  albumin. 

After  the  administration  of  certain  drugs  a  white  precipitate  of 
resin  acids  may  form  at  the  point  of  contact  of  the  two  fluids  and 
may  cause  the  observer  to  draw  wrong  conclusions.  This  ring,  if 
composed  of  resin  acids,  will  dissolve  in  alcohol,  whereas  the  albu- 
min ring  will  not  dissolve. 

Weinberger  has  recently  shown  that  a  ring  closely  resembling  the 
albumin  ring  is  often  obtained  in  urines  preserved  by  thymol  when 
subjected  to  Heller's  test.  The  ring  is  due  to  the  formation  of  nitro- 
sothymol  and  possibly  nitrothymol.  If  the  thymol  is  removed  from 
the  urine  by  extraction  with  petroleum  ether1  previous  to  adding 
nitric  acid,  the  ring  does  not  form. 

An  instrument  called  the  albunioscope  (horismascope)  has  been 
devised  for  use  in  this  test  and  has  met  with  considerable  favor. 
The  method  of  using  the  albumoscope  is  described  below. 

Use  of  the  Albunioscope. — This  instrument  is  intended  to  facili- 
tate the  making  of  "  ring  "  tests  such  as  Heller's  and  Roberts'.  In 
making  a  test  about  5  c.c.  of  the  solution  under  examination  is  first 
introduced  into  the  apparatus  through  the  larger  arm  and  the  re- 
agent used  in  the  particular  test  is  then  introduced  through  the 
capillary  arm  and  allowed  to  flow  down  underneath  the  solution 
under  examination.  If  a  reasonable  amount  of  care  is  taken  there 
is  no  possibility  of  mixing  the  two  solutions  and  a  definitely  defined 
white  "  ring  "  is  easily  obtained  at  the  zone  of  contact. 

2.  Roberts'  Ring  Test. — Place  5  c.c.  of  Roberts'  reagent-  in  a 
test-tube,  incline  the  tube,  and,  by  means  of  a  pipette,  allow  the 
urine  to  flow  slowly  down  the  side.  The  liquids  should  stratify 
with  the  formation  of  a  white  zone  of  precipitated  albumin  at  the 
point  of  juncture.  This  test  is  a  modification  of  Heller's  ring  test 
and  is  rather  more  satisfactory  than  that  test,  since  the  colored  rings 
never  form  and  the  consequent  confusion  is  avoided.  The  albu- 
moscope (see  above)  may  also  be  used  in  making  this  test. 

Accomplished  readily  by  gently  agitating  equal  volumes  of  petroleum  ether 
and  the  urine  under  examination,  for  tzvo  minutes  in  a  test-tube,  before  ap- 
plying the  test. 

"  Roberts'  reagent  is  composed  of  1  volume  of  concentrated  HXOj  and  5 
volumes  of  a  saturated   solution  of  MgSO.. 


316  PHYSIOLOGICAL    CHEMISTRY. 

3.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent1  in 
a  test-tube,  incline  the  tube,  and,  by  means  of  a  pipette,  allow  5  c.c. 
of  urine,  acidified  with  acetic  acid,  to  flow  slowly  down  the  side.  A 
white  zone  will  form  at  the  point  of  contact.  This  is  an  exceedingly- 
delicate  test,  in  fact,  too  delicate  for  ordinary  clinical  purposes, 
since  it  serves  to  detect  albumin  when  present  in  the  merest  trace 
(1  1250,000)  and  hence  most  normal  urines  will  give  a  positive  re- 
action for  albumin  when  this  test  is  applied. 

Some  investigators  claim  that  the  delicacy  of  this  test  depends 
upon  the  presence  of  sodium  chloride  in  the  urine,  the  test  losing 
accuracy  if  the  sodium  chloride  content  be  low. 

4.  Jolles'  Reaction. — Shake  5  c.c.  of  urine  with  1  c.c.  of  30  per 
cent  acetic  acid  and  4  c.c.  of  Jolles'  reagent2  in  a  test-tube.  A  white 
precipitate  indicates  the  presence  of  albumin. 

Care  should  be  taken  to  use  the  correct  amount  of  acetic  acid, 
since  the  use  of  too  small  an  amount  may  result  in  the  formation  of 
mercury  combinations  which  may  cause  confusion.  In  the  presence 
of  iodine,  mercuric  iodide  will  form  but  may  readily  be  differenti- 
ated from  albumin  through  the  fact  that  it  is  soluble  in  alcohol. 

5.  Coagulation  or  Boiling  Test. —  (a)  Heat  5  c.c.  of  urine  to 
boiling  in  a  test-tube.  A  precipitate  forming  at  this  point  is  due 
either  to  albumin  or  to  phosphates.  Acidify  the  urine  slightly  by 
the  addition  of  3-5  drops  of  very  dilute  acetic  acid,  adding  the  acid 
drop  by  drop  to  the  hot  solution.  If  the  precipitate  is  due  to  phos- 
phates it  will  disappear  under  these  conditions,  whereas  if  it  is  due 
to  albumin  it  will  not  only  fail  to  disappear  but  will  become  more 
flocculent  in  character,  since  the  reaction  of  a  fluid  must  be  acid  to 
secure  the  complete  precipitation  of  the  albumin  by  this  coagulation 
process.  Too  much  acid  should  be  avoided  since  it  will  cause  the 
albumin  to  go  into  solution.  Certain  resin  acids  may  be  precipi- 
tated by  the  acid,  but  the  precipitate  due  to  this  cause  may  be  easily 
differentiated  from  the  albumin  precipitate  by  reason  of  its  solubility 
in  alcohol. 

1  Spiegler's  reagent  has  the  following  composition : 

Tartaric    acid 20  grams. 

Mercuric  chloride 40  grams. 

Glycerol    100  grams. 

Distilled  water 1000  grams. 

2  Jolles'  reagent  has  the  following  composition  : 

Succinic    acid 40  grams. 

Mercuric  chloride 20  grams. 

Sodium    chloride 20  grams. 

Distilled  water   1000  grams. 


URINE.  317 

(h)  A  modification  of  this  test  in  quite  general  use  is  as  follows: 
Fill  a  test-tube  two-thirds  full  of  urine  and  gently  heal  the  upper 
half  of  the  fluid  to  boiling,  being  careful  that  this  fluid  does  nol  mix 
with  the  lower  half.     A  turbidity  indicates  albumin  or  phosph 

Acidify  the  urine  slightly  by  the  addition  of  3  5  drops  of  dilute 
acetic  acid,  when  the  turbidity,  if  due  to  phosphates,  will  disap- 
pear. 

Nitric  acid  is  often  used  in  place  of  acetic  acid  in  these  t c - 1 > .  In 
case  nitric  acid  is  used  ordinarily  1—2  drops  is  sufficient. 

6.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c. 
of  urine  in  a  test-tube  add  5—10  drops  of  acetic  acid.  Mix  well  and 
add  potassium  ferrocyanide  drop  by  drop,  until  a  precipitate  forms. 
This  is  a  very  delicate  test.  Schmiedl  claims  that  a  precipitate  of 
Fe(Cn)cK2Zn  or  Fe(Cn)cZn2  is  formed  when  urines  contain- 
ing zinc  are  subjected  to  this  test  and  that  this  precipitate  resembles 
the  precipitate  secured  with  protein  solutions.  In  the  case  of 
human  urine  a  reaction  was  obtained  when  0.000022  gram  of  zinc 
per  cubic  centimeter  was  present.  Schmiedl  further  found  that  the 
urine  collected  from  rabbits  housed  in  zinc-lined  cages  possessed  a 
zinc  content  which  was  sufficient  to  yield  a  ready  response  to  the 
test.    Zinc  is  the  only  interfering  substance  so  far  reported. 

7.  Tanret's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  Tanret's 
reagent1  drop  by  drop  until  a  turbidity  or  precipitate  forms.  This 
is  an  exceedingly  delicate  test.  Sometimes  the  urine  is  stratified 
upon  the  reagent  as  in  Heller's  or  Roberts'  ring  test.  According 
to  Repiton,  urates  interfere  with  the  delicacy  of  this  test.  Tanret. 
however,  claims  that  urates  do  not  interfere  inasmuch  as  any  pre- 
cipitate due  to  urates  may  be  brought  into  solution  by  heat  whereas 
an  albumin  precipitate  under  the  same  conditions  will  persist. 
Tanret  further  states  that  mucin  interferes  with  the  delicacy  of  the 
test  and  that  it  should  therefore  be  removed  from  the  urine  under 
examination,  by  acidification  with  acetic  acid  and  filtration  before 
testing  for  albumin. 

8.  Sodium  Chloride  and  Acetic  Acid  Test. — Mix  two  volumes 
of  urine  and  one  volume  of  a  saturated  solution  of  sodium  chloride 
in  a  test-tube,  acidify  with  acetic  acid  and  heat  to  boiling.  The  pro- 
duction of  a  cloudiness  or  the  formation  of  a  precipitate  indicates 

tanret's  reagent  is  prepared  as  follows:  Dissolve  [.35  gram  of  mercuric 
chloride  in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide 
dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c.  with  water 
and  add  20  c.c.  of  glacial   acetic  acid  to  the  mixture. 


3  l8  PHYSIOLOGICAL    CHEMISTRY. 

the  presence  of  albumin.  The  resin  acids  may  interfere  here  as  in 
the  ordinary  coagulation  test  (page  316)  but  they  may  be  easily 
differentiated  from  albumin  by  means  of  their  solubility  in  alcohol. 

GLOBULIN. 

Serum  globulin  is  not  a  constituent  of  normal  urine  but  fre- 
quently occurs  in  the  urine  under  pathological  conditions  and  is 
ordinarily  associated  with  serum  albumin.  In  albuminuria  globulin 
in  varying  amounts  often  accompanies  the  albumin,  and  the  clinical 
significance  of  the  two  is  very  similar.  Under  certain  conditions 
globulin  may  occur  in  the  urine  unaccompanied  by  albumin. 

.    Experiments. 

Globulin  will  respond  to  all  the  tests  just  outlined  under  Albumin. 
If  it  is  desirable  to  differentiate  between  albumin  and  globulin  in 
any  urine  the  following  processes  may  be  employed : 

1.  Saturation  With  Magnesium  Sulphate. — Place  25  c.c.  of 
neutral  urine  in  a  small  beaker  and  add  pulverized  magnesium  sul- 
phate in  substance  to  the  point  of  saturation.  If  the  protein  present 
is  globulin  it  will  precipitate  at  this  point.  If  no  precipitate  is  pro- 
duced acidify  the  saturated  solution  with  acetic  acid  and  warm 
gently.     Albumin  will  be  precipitated  if  present. 

The  above  procedure  may  be  used  to  separate  globulin  and  albu- 
min if  present  in  the  same  urine.  To  do  this  filter  off  the  globulin 
after  it  has  been  precipitated  by  the  magnesium  sulphate,  then 
acidify  the  clear  solution  and  warm  gently  as  directed.  Note  the 
formation  of  the  albumin  precipitate. 

2.  Half-Saturation  With  Ammonium  Sulphate. — Place  25  c.c. 
of  neutral  urine  in  a  small  beaker  and  add  an  equal  volume  of  a 
saturated  solution  of  ammonium  sulphate.  Globulin,  if  present, 
will  be  precipitated.  If  no  precipitate  forms  add  ammonium  sul- 
phate in  substance  to  the  point  of  saturation.  If  albumin  is  present 
it  will  be  precipitated  upon  saturation  of  the  solution  as  just  indi- 
cated. This  method  may  also  be  used  to  separate  globulin  and 
albumin  when  they  occur  in  the  same  urine. 

Frequently  in  urine  which  contains  a  large  amount  of  urates  a 
precipitate  of  ammonium  urate  may  occur  when  the  ammonium 
sulphate  solution  is  added  to  the  urine.  This  urate  precipitate 
should  not  be  confounded  with  the  precipitate  due  to  globulin. 
The  two  precipitates  may  be  differentiated  by  means  of , the  fact  that 
the  urate  precipitate  ordinarily  appears  only  after,  the  lapse  of  sev- 
eral minutes  whereas  the  globulin  generally  precipitates  at  once. 


URINE.  319 

PROTEOSE  AND  PEPTONE. 

Proteoses,  particularly  deutero-proteose  and  hetero  proteose,  have 
frequently  been  found  in  the  urine  under  various  pathological  con- 
ditions such  as  diphtheria,  pneumonia,  intestinal  ulcer,  carcinoma, 
dermatitis,  osteomalacia,  atrophy  of  the  kidneys  and  in  sarcomata 
of  the  bones  of  the  trunk.  "  Bence-Jones'  protein,"  a  proteose-like 
substance,  is  of  interest  in  this  connection  and  its  appearance  in  the 
urine  is  believed  to  be  of  great  diagnostic  importance  in  cases  of 
multiple  myeloma  or  myelogenic  osteosarcoma.  By  some  investi- 
gators this  protein  is  held  to  be  a  variety  of  hetero-proteose  whereas 
others  claim  that  it  possesses  albumin  characteristics. 

Peptone  certainly  occurs  much  less  frequently  as  a  constituent  of 
the  urine  than  does  proteose,  in  fact  most  investigators  seriously 
question  its  presence  under  any  conditions.  There  are  many  in- 
stances of  peptonuria  cited  in  the  early  literature  but  because  of  the 
uncertainty  in  the  conception  of  what  really  constituted  a  peptone 
it  is  probable  that  in  many  cases  of  so-called  peptonuria  the  protein 
present  was  really  proteose. 

Experiments. 

1.  Boiling  Test. — Make  the  ordinary  coagulation  test  according 
to  the  directions  given  under  Albumin,  page  316.  If  no  coagulable 
protein  is  found  allow  the  boiled  urine  to  stand  and  note  the  gradual 
appearance,  in  the  cooled  fluid,  of  a  flaky  precipitate  of  proteose. 
This  is  a  crude  test  and  should  never  be  relied  upon. 

2.  Schulte's  Method. — Acidify  50  c.c.  of  urine  with  dilute  acetic 
acid  and  filter  off  any  precipitate  of  nucleoprotein  which  may  form. 
Now  test  a  few  cubic  centimeters  of  the  urine  for  coagulable  pro- 
tein, by  tests  2  and  5  under  Albumin,  pp.  315  and  316.  If  coagula- 
ble protein  is  present  remove  it  by  coagulation  and  filtration  before 
proceeding.  Introduce  25  c.c.  of  the  urine,  freed  from  coagulable 
protein,  into  150  c.c.  of  absolute  alcohol  and  allow  it  to  stand  for 
12-24  hours.  Decant  the  supernatant  fluid  and  dissolve  the  pre- 
cipitate in  a  small  amount  of  hot  water.  Now  filter  this  solution, 
and  after  testing  again  for  nucleoprotein  with  very  dilute  acetic 
acid,  try  the  biuret  test.  If  this  test  is  positive  the  presence  of  pro- 
teose is  indicated.1 

Urobilin  does  not  ordinarily  interfere  with  this  test  since  it  is  al- 
most entirely  dissolved  by  the  absolute  alcohol  when  the  proteose 
is  precipitated. 

'If  it  is  considered  desirable  to  test  for  peptone  the  proteose  may  he  removed 
by  saturation  with  (NH.hSCX  according'  to  the  directions  given  on  page  115  and 
the  filtrate  tested  for  peptone  by  the  biuret  test. 


320  PHYSIOLOGICAL    CHEMISTRY. 

3.  v.  Aldor's  Method. — Acidify  10  c.c.  of  urine  with  hydro- 
chloric acid,  add  phosphotungstic  acid  until  no  more  precipitate 
forms  and  centifugate1  the  solution.  Decant  the  supernatant  fluid, 
add  some  absolute  alcohol  to  the  precipitate  and  centrifugate  again. 
This  washing  with  alcohol  is  intended  to  remove  the  urobilin  and 
hence  should  be  continued  so  long  as  the  alcohol  exhibits  any  colora- 
tion whatever.  Now  suspend  the  precipitate  in  water  and  add 
potassium  hydroxide  to  bring  it  into  solution.  At  this  point  the 
solution  may  be  blue  in  color,  in  which  case  decolorization  may  be 
secured  by  gently  heating.  Apply  the  biuret  test  to  the  cool  solu- 
tion.    A  positive  biuret  test  indicates  the  presence  of  proteoses. 

4.  Detection  of  "  Bence-Jones'  Protein." — Heat  the  suspected 
urine  very  gently,  carefully  noting  the  temperature.  At  as  low  a 
temperature  as  40 °  C.  a  turbidity  may  be  observed  and  as  the  tem- 
perature is  raised  to  about  60 °  C.  a  flocculent  precipitate  forms  and 
clings  to  the  sides  of  the  test-tube.  If  the  urine  is  now  acidified 
very  slightly  with  acetic  acid  and  the  temperature  further  raised  to 
ioo°  C.  the  precipitate  at  least  partly  disappears;  it  will  return  upon 
cooling  the  tube. 

This  property  of  precipitating  at  so  low  a  temperature  and  of 
dissolving  at  a  higher  temperature  is  typical  of  "  Bence-Jones'  pro- 
tein" and  may  be  used  to  differentiate  it  from  all  other  forms  of 
protein  material  occurring  in  the  urine. 

NUCLEOPROTEIN. 

There  has  been  considerable  controversy  as  to  the  proper  classi- 
fication for  the  protein  body  which  forms  the  "  nubecula "  of 
normal  urine.  By  different  investigators  it  has  been  called  mucin, 
mucoid,  phospho protein,  nude 0 albumin  and  nude o protein.  Of 
course,  according  to  the  modern  acceptation  of  the  meanings  of 
these  terms  they  cannot  be  synonymous.  Mucin  and  mucoid  are  gly- 
coproteins and  hence  contain  no  phosphorus  (see  p.  106),  whereas 
phosphoproteins  and  nucleoproteins  are  phosphorized  bodies.  It 
may  possibly  be  that  both  these  forms  of  protein,  i.  e.,  the  glycopro- 
tein and  the  phosphorized  type,  occur  in  the  urine  under  certain  con- 
ditions (see  page  290).  In  this  connection  we  will  use  the  term 
nucleo protein.  The  pathological  conditions  under  which  the  con- 
tent of  nucleoprotein  is  increased  includes  all  affections  of  the 
urinary  passages  and  in  particular  pyelitis,  nephritis  and  inflamma- 
tion of  the  bladder. 

1  If  not  convenient  to  use  a  centrifuge  the  precipitate  may  be  filtered  off  and 
washed  on  the  filter  paper  with  alcohol. 


URINE.  32  1 

Experiments. 

t.   Detection  of  Nucleoprotein. —  Place    [0  c.c.   of  urine  in  a 
small  beaker,  dilute  it  with  three  volumes  of  water,  to  prevenl  pre 
cipit'ation  of  urates,  and  make  the  reaction  very  strongly  acid  with 
acetic  acid.      If    the  urine    becomes  turbid  it  is  an  indication  that 
nucleoprotein  is  present. 

If  the  urine  under  examination  contains  albumin  the  greater  por- 
tion of  this  substance  should  be  removed  by  boiling  the  urine  before 
testing  it  for  the  presence  of  nucleoprotein. 

2.  Ott's  Precipitation  Test. — Mix  25  c.c.  of  the  urine  with  an 
equal  volume  of  a  saturated  solution  of  sodium  chloride  and  slowly 
add  Almen's  reagent.1  In  the  presence  of  nucleoprotein  a  volumin- 
ous precipitate  forms. 

BLOOD. 

The  pathological  conditions  in  which  blood  occurs  in  the  urine 
may  be  classified  under  the  two  divisions  hematuria  and  hemo- 
globinuria. In  haematuria  we  are  able  to  detect  not  only  the  haemo- 
globin but  the  unruptured  corpuscles  as  well,  whereas  in  hemo- 
globinuria the  pigment  alone  is  present.  Hematuria  is  brought 
about  through  blood  passing  into  the  urine  because  of  some  lesion  of 
the  kidney  or  of  the  urinary  tract  below  the  kidney.  Hemoglobi- 
nuria is  brought  about  through  hemolysis,  i.  e.,  the  rupturing  of 
the  stroma  of  the  erythrocyte  and  the  liberation  of  the  haemoglobin. 
This  may  occur  in  scurvy,  typhus,  pyemia,  purpura  and  in  other 
diseases.  It  may  also  occur  as  the  result  of  a  burn  covering  a  con- 
siderable area  of  the  body,  or  may  be  brought  about  through  the 
action  of  certain  poisons  or  by  the  injection  of  various  substances 
having  the  power  of  dissolving  the  erythrocytes.  Transfusion  of 
blood  may  also  cause  hemoglobinuria. 

Experiments. 

1.  Heller's  Test. — Render  10  c.c.  of  urine  strongly  alkaline  with 
potassium  hydroxide  solution  and  heat  to  boiling.  Upon  allowing 
the  heated  urine  to  stand  a  precipitate  of  phosphates,  colored  red  by 
the  contained  hematin,  is  formed.  It  is  ordinarily  well  to  make  a 
"  control  "  experiment  using  normal  urine,  before  coming  to  a  final 
decision. 

Certain  substances  such  as  cascara  sagrada,   rhubarb,  santonin, 

1  Dissolve  5  grams  of  tannin  in  240  c.c.  of  50  per  cent  alcohol  and  add  10  c.c. 
of- 25  per  cent  acetic  acid. 
22 


322  PHYSIOLOGICAL    CHEMISTRY. 

and  senna  cause  the  urine  to  give  a  similar  reaction.  Reactions  due 
to  such  substances  may  be  differentiated  from  the  true  blood  reac- 
tion by  the  fact  that  both  the  precipitate  and  the  pigment  of  the 
former  reaction  disappear  when  treated  with  acetic  acid,  whereas  if 
the  color  is  due  to  hsematin  the  acid  will  only  dissolve  the  precipitate 
of  phosphates  and  leave  the  pigment  undissolved. 

2.  Teichmann's  Haemin  Test. — Place  a  small  drop  of  the  sus- 
pected urine  or  a  small  amount  of  the  moist  sediment  on  a  micro- 
scopic slide,  add  a  minute  grain  of  sodium  chloride  and  carefully 
evaporate  to  dryness  over  a  low  flame.  Put  a  cover  glass  in  place, 
run  underneath  it  a  drop  of  glacial  acetic  acid  and  warm  gently 
until  the  formation  of  gas  bubbles  is  observed.  Cool  the  prepara- 
tion, examine  under  the  microscope  and  compare  the  form  of  the 
crystals  with  those  reproduced  in  Figs.  58  and  59,  page  198.  (See 
Atkinson  and  Kendall's  modification,  p.  197.) 

3.  Heller-Teichmann  Reaction. — Produce  the  pigmented  pre- 
cipitate according  to  directions  given  in  Heller's  test  on  p.  321.  If 
there  is  a  copious  precipitate  of  phosphates  and  but  little  pigment 
the  phosphates  may  be  dissolved  by  treatment  with  acetic  acid  and 
the  residue  used  in  the  formation  of  the  haemin  crystals  according 
to  directions  in  Experiment  2,  above. 

4.  v.  Zeynek  and  Nencki's  Haemin  Test. — To  10  c.c.  of  the 
urine  under  examination  add  acetone  until  no  more  precipitate 
forms.  Filter  off  the  precipitate  and  extract  it  with  10  c.c.  of 
acetone  rendered  acid  with  2-3  drops  of  hydrochloric  acid.  Place 
a  drop  of  the  resulting  colored  extract  on  a  slide,  immediately  place 
a  cover  glass  in  position  and  examine  under  the  microscope.  Com- 
pare the  form  of  the  crystals  with  those  shown  in  Figs.  58  and  59, 
page  198.  Haemin  crystals  produced  by  this  manipulation  are 
sometimes  very  minute,  thus  rendering  it  difficult  to  determine  the 
exact  form  of  the  crystal. 

5.  Schalfijew's  Haemin  Test. — Place  20  c.c.  of  glacial  acetic 
acid  in  a  small  beaker  and  heat  to  8o°  C.  Add  5  c.c.  of  the  urine 
under  examination,  raise  the  temperature  to  8o°  C.  and  stand  the 
mixture  aside  to  cool.  Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  shown  in  Figs.  58  and  59,  page  198. 

6.  Guaiac  Test. — Place  5  c.c.  of  urine  in  a  test-tube  and  by 
means  of  a  pipette  introduce  a  freshly  prepared  alcoholic  solution 
of  guaiac  (strength  about  1  :6o)  into  the  fluid  until  a  turbidity  re- 
sults then  add  old  turpentine  or  hydrogen  peroxide,  drop  by  drop, 
until  a  blue  color  is  obtained.     This  is  a  very  delicate  test  when 


URINE.  323 

properly  performed.      Buckmaster  has  recently  suggested  the  use 

of  guaiaconic  acid  instead  of  the  solution  of  guaiac.      Sec  discussion 
on  page  192  and  test  on  page  T96. 

7.  Schumm's  Modification  of  the  Guaiac  Test.— To  about  5 
c.c.  of  urine1  in  a  test-tube  add  about  10  drop-  of  a  freshly  prepared 
alcoholic  solution  of  guaiac.  Agitate  the  tube  gently,  add  about  20 
drops  of  old  turpentine,  subject  the  tube  to  a  thorough  shaking  and 
permit  it  to  stand  for  about  2-3  minutes.  A  blue  color  indicates 
the  presence  of  blood  in  the  solution  under  examination.  In  case 
there  is  insufficient  blood  to  yield  a  blue  color  under  these  con 
ditions,  a  few  c.c.  of  alcohol  should  be  added  and  the  tube  gently 
shaken,  whereupon  a  blue  coloration  will  appear  in  the  upper  al- 
cohol-turpentine layer. 

A  control  test  should  always  be  made  using  water  in  place  of 
urine.  In  the  detection  of  very  minute  traces  of  blood  only  3-5 
drops  of  the  guaiac  solution  should  be  employed. 

8.  Adler's  Benzidine  Reaction. — This  is  one  of  the  most  deli- 
cate of  the  reactions  for  the  detection  of  blood.  Different  benzidine 
preparations  vary  greatly  in  their  sensitiveness,  however.  Inas- 
much as  benzidine  solutions  change  readily  upon  contact  with  light, 
it  is  essential  that  they  be  kept  in  a  dark  place.  The  test  is  per- 
formed as  follows:  To  a  saturated  solution  of  benzidine  in  alcohol 
or  glacial  acetic  acid  add  an  equal  volume  of  3  per  cent  hydrogen 
peroxide  and  one  c.c.  of  the  urine  under  examination.  If  the  mix- 
ture is  not  already  acid,  render  it  so  with  acetic  acid,  and  note  the 
appearance  of  a  green  or  blue  color.  A  control  test  should  be  made 
substituting  water  for  the  urine. 

Often  when  urines  containing  a  small  amount  of  blood  are  tested 
by  this  reaction,  the  mixture  is  rendered  so  turbid  as  to  make  it 
difficult  to  decide  as  to  the  presence  of  a  faint  green  color.  Such 
urines  should  be  extracted  with  an  ether-acetic  acid  solution  and 
the  resulting  extract  washed  with  water  before  the  test  is  applied 
to  it.  The  sensitiveness  of  the  benzidine  reaction  is  greater  when 
applied  to  aqueous  solutions  than  when  applied  to  the  urine. 

9.  Spectroscopic  Examination. — Submit  the  urine  to  a  spectro- 
scopic examination  according  to  the  directions  given  on  page  203 
looking  especially  for  the  absorption-bands  of  oxyhannoglobin  and 
methsemoglobin  (see  Absorption  Spectra,  Plate  I.  I. 

1  Alkaline  urine  should  be  made  slightly  acid  with  acetic  acid  as  the  blue  end- 
reaction  is  very  sensitive  to  alkali. 


324  PHYSIOLOGICAL    CHEMISTRY. 

BILE. 

Both  the  pigments  and  the  acids  of  the  bile  may  be  detected 
in  the  urine  under  certain  pathological  conditions.  Of  the  pig- 
ments, bilirubin  is  the  only  one  which  has  been  positively  identi- 
fied in  fresh  urine ;  the  other  pigments,  when  present,  are  probably 
derived  from  the  bilirubin.  A  urine  containing  bile  may  be  yel- 
lowish-green to  brown  in  color  and  when  shaken  foams  readily. 
The  staining  of  the  various  tissues  of  the  body  through  the  ab- 
sorption of  bile  due  to  occlusion  of  the  bile  duct  causes  a  condi- 
tion known  as  icterus  or  jaundice.  Bile  is  always  present  in  the 
urine  under  such  conditions  unless  the  amount  of  bile  reaching 
the  tissues  is  extremely  small. 

Experiments. 

Tests  for  Bile  Pigments. 

1.  Gmelin's  Test. — To  about  5  c.c.  of  concentrated  nitric  acid 
in  a  test-tube  add  an  equal  volume  of  urine  carefully  so  that  the 
two  fluids  do  not  mix.  At  the  point  of  contact  note  the  various 
colored  rings,  green,  blue,  violet,  red  and  reddish-yellow. 

2.  Rosenbach's  Modification  of  Gmelin's  Test. — Filter  5  c.c. 
of  urine  through  a  small  filter  paper.  Introduce  a  drop  of  con- 
centrated nitric  acid  into  the  cone  of  the  paper  and  observe  the 

■  succession  of  colors  as  given  in  Gmelin's  test. 

3.  Nakayama's  Reaction. — To  5  c.c.  of  urine  in  a  test-tube  add 
an  equal  volume  of  a  10  per  cent  solution  of  barium  chloride.  Cen- 
trifugate  the  mixture,  pour  off  the  supernatant  fluid  and  heat  the 
precipitate  with  2  c.c.  of  Nakayama's  reagent.1  In  the  presence 
of  bile  pigments  the  solution  assumes  a  blue  or  green  color. 

3.  Huppert's  Reaction. — Thoroughly  shake  equal  volumes  of 
urine  and  milk  of  lime  in  a  test-tube.  The  pigments  unite  with 
the  calcium  and  are  precipitated.  Filter  off  the  precipitate,  wash 
it  with  water  and  transfer  to  a  small  beaker.  Add  alcohol  acidified 
slightly  with  hydrochloric  acid  and  warm  upon  a  water-bath  until 
the  solution  becomes  colored  an  emerald  green. 

According  to  Steensma,  this  procedure  may  give  negative  results 
even  in  the  presence  of  the  pigments,  owing  to  the  fact  that  the 
acid-alcohol  is  not  a  sufficiently  strong  oxidizing  agent.  He  there- 
fore suggests  the  addition  of  a  drop  of  a  0.5  per  cent  solution  of 

1  Prepared  by  combining  99  c.c.  of  alcohol  and  1  c.c.  of  fuming  hydrochloric 
acid  containing  4  grams  of  ferric  chloride  per  liter. 


urim:.  325 

sodium  nitrite  to  the  acid-alcohol  mixture  before  warming  on  the 

water-bath.     Try  this  modification  also. 

5.  Salkowski's  Test.— Render  5  c.c.  of  urine  alkaline  with  a 
few  drops  of  a  10  per  cent  sodium  carbonate  solution  and  add  a 
10  per  cent  solution  of  calcium  chloride,  drop  by  drop,  until   the 

supernatant  fluid  exhibits  the  normal  urinary  color  when  the  con- 
tents of  the  test-tube  are  thoroughly  mixed.  Filter  off  the  pre- 
cipitate, and  after  washing  it,  place  it  in  a  second  tube  with  95 
per  cent  alcohol.  Acidify  the  alcohol  with  hydrochloric  acid  and, 
if  necessary,  shake  the  tube  to  bring  the  precipitate  into  solution. 
Heat  the  solution  to  boiling  and  observe  the  appearance  of  a  green 
color  which  changes  through  blue  and  violet  to  red;  if  no  bile  is 
present  the  solution  does  not  undergo  any  color  change.  This  test 
will  frequently  exhibit  greater  delicacy  than  Gmelin's  test.  Steens- 
ma's  suggestions  mentioned  under  Huppert's  Reaction,  p.  324,  ap- 
ply in  connection  with  this  test  also. 

4.  Hammarsten's  Reaction. — To  about  5  c.c.  of  Hammarsten- 
reagent1  in  a  small  evaporating  dish  add  a  few  drops  of  urine.  A 
green  color  is  produced.  If  more  of  the  reagent  is  now  added  the 
play  of  colors  as  noted  in  Gmelin's  test  may  be  obtained. 

5.  Smith's  Test. — To  2-3  c.c.  of  urine  in  a  test-tube  add  care- 
fully about  5  c.c.  of  dilute  tincture  of  iodine  (1  :  10)  so  that  the 
fluids  do  not  mix.  A  green  ring  is  observed  at  the  point  of  con- 
tact. 

6.  Salkowski-Schippers  Reaction. — Neutralize  the  aciditv  of 
10  c.c.  of  the  urine  under  examination  with  a  few  drops  of  a 
dilute  solution  of  sodium  carbonate,  and  add  5  drops  of  a  20  per 
cent  solution  of  sodium  carbonate  and  10  drops  of  a  20  per  cent 
solution  of  calcium  chloride.  Filter  off  the  resultant  precipitate 
upon  a  hardened  filter-paper  and  wash  it  with  water.  Remove 
the  precipitate  to  a  small  porcelain  dish,  add  3  c.c.  of  an  acid- 
alcohol  mixture2  and  a  few  drops  of  a  dilute  solution  of  sodium 
nitrite  and  heat.  The  production  of  a  green  color  indicates  the 
presence  of  bile  pigments. 

Tests  for  Bile  Acids. 

i.  Pettenkofer's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  5 
drops  of  a  5  per  cent  solution  of  sucrose.     Now  incline  the  tube. 

1  Hammarsten's  reagent  is  made  by  mixing  I  volume  of  25  per  cent  nitric  acid 
and  19  volumes  of  25  per  cent  hydrochloric  acid  and  then  adding  1  volume  oi  this 
acid  mixture  to  4  volumes  of  95  per  cent  alcohol. 

2  Made  by  adding  5  c.c.  of  concent  rated  hydrochloric  acid  to  05  c.c.  of  06  per 
cent  alcohol. 


326  PHYSIOLOGICAL    CHEMISTRY. 

run  about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down 
the  side  and  note  the  red  ring  at  the  point  of  contact.  Upon  slightly 
agitating  the  contents  of  the  tube  the  whole  solution  gradually  as- 
sumes a  reddish  color.  As  the  tube  becomes  warm,  it  should  be 
cooled  in  running  water  in  order  that  the  temperature  may  not 
rise  above  70  °  C. 

2.  Mylius's  Modification  of  Pettenkofer's  Test. — To  approx- 
imately 5  c.c.  of  urine  in  a  test-tube  add  3  drops  of  a  very  dilute 
(1:1,000)    aqueous  solution  of  furfurol, 

HC— CH 

HC        C-CHO. 
\/ 
0 

Now  incline  the  tube,  run  about  2-3  c.c.  of  concentrated  sulphuric 
acid  carefully  down  the  side  and  note  the  red  ring  as  above.  In 
this  case  also,  upon  shaking  the  tube,  the  whole  solution  is  colored 
red.     Keep  the  temperature  below  70  °  C.  as  before. 

3.  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few 
drops  of  urine  in  an  evaporating  dish  add  a  trace  of  a  dilute 
sucrose  solution  and  one  or  more  drops  of  dilute  sulphuric  acid. 
Evaporate  on  a  water-bath  and  observe  the  development  of  a  violet 
color  at  the  edge  of  the  evaporating  mixture.  Discontinue  the 
evaporation  as  soon  as  the  color  is  observed. 

4.  v.  Udransky's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add 
3-4  drops  of  a  very  dilute  (1  :  1,000)  aqueous  solution  of  furfurol. 
Place  the  thumb  over  the  top  of  the  tube  and  shake  until  a  thick 
foam  is  formed.  By  means  of  a  small  pipette  add  2-3  drops  of  con- 
centrated sulphuric  acid  to  the  foam  and  observe  the  dark  pink  col- 
oration produced. 

6.  Hay's  Test. — This  test  is  based  upon  the  principle  that  bile 
acids  have  the  property  of  reducing  the  surface  tension  of  fluids 
in  which  they  are  contained.  The  test  is  performed  as  follows : 
Cool  about  10  c.c.  of  urine  in  a  test-tube  to  170  C.  or  lower,  and 
sprinkle  a  little  finely  pulverized  sulphur  upon  the  surface  of  the 
fluid.  The  presence  of  bile  acids  is  indicated  if  the  sulphur  sinks 
to  the  bottom  of  the  liquid,  the  rapidity  with  which  the  sulphur 
sinks  depending  upon  the  amount  of  bile  acids  present  in  the  urine. 
The  test  is  said  to  react  with  bile  acids  when  the  latter  are  present 
in  the  proportion  1  :  120,000. 


URINE.  327 

Some  investigators  claim  that   it   is  impossible  to  differentiate 
between  bile  acids  and  bile  pigments  by  this  test. 

(II.. 


ACETONE,   (  !  =  0. 
I  I 


L 


it  was  formerly  very  generally  believed  that  acetone  appeared  111 
the  urine  under  pathological  conditions  because  of  increased  protein 
decomposition.  It  is  now  generally  thought  that,  in  man,  the  nut- 
put  of  acetone  arises  principally  from  the  breaking  down  of  fatty 
tissues  or  fatty  foods  within  the  organism.  The  quantity  of  acetone 
eliminated  has  been  shown  to  increase  when  the  subject  is  fed  an 
abundance  of  fat-containing  food  as  well  as  during  fasting,  where- 
as a  replacement  of  the  fat  with  carbohydrates  is  followed  by  a 
marked  decrease  in  the  acetone  excretion.  Conditions  are  different 
with  certain  of  the  lower  animals.  With  the  dog,  for  instance, 
the  output  of  acetone  is  not  diminished  when  the  animal  is  fed 
upon  a  carbohydrate  diet,  is  decreased  during  fasting  and  increased 
when  the  animal  is  fed  upon  a  diet  of  meat. 

Acetone  and  the  closely  related  bodies,  /3-oxybutyric  acid  and  dia- 
cetic  acid,  are  generally  classified  as  the  acetone  bodies.  They  are 
all  associated  with  a  deranged  metabolic  function  and  may  appear 
in  the  urine  together  or  separately,  depending  upon  the  conditions. 
Acetone  and  diacetic  acid  may  occur  alone  in  the  urine  but  |8-oxy- 
butyric  acid  is  never  found  except  in  conjunction  with  one  or  the 
other  of  these  bodies.  Acetone  and  diacetic  acid  arise  chiefly  from 
the  oxidation  of  /8-oxybutyric  acid.  The  relation  existing  between 
these  three  bodies  is  shown  in  the  following  reactions : 

(a)  CH3CH(OH)CH2-COOH  +  0  = 

^3-oxybutyric  acid. 

CH,COCH2COOH  +  11,0. 

Diacetic  acid. 

(b)  CH3CO-CH2-COOH=  (CH3)2CO  +  CO,. 

Diacetic  acid.  Acetone. 

Acetone,  chemically  considered,  is  a  ketone,  di-methyl  ketone. 
When  pure  it  is  a  liquid  which  possesses  a  characteristic  aromatic 
fruit-like  odor,  boils  at  56— 570  C.  and  is  miscible  with  water,  alcohol 
or  ether  in  all  proportions.     Acetone  is  a  physiological  as  well  as 


328  PHYSIOLOGICAL    CHEMISTRY. 

a  pathological   constituent   of   the  urine   and   under   normal   con- 
ditions the  daily  output  is  about  0.01-0.03  gram. 

Pathologically,  the  elimination  of  acetone  is  often  greatly  in- 
creased and  at  such  times  a  condition  of  acetonuria  is  said  to  exist. 
This  pathological  acetonuria  may  accompany  diabetes  mellitus, 
scarlet  fever,  typhoid  fever,  pneumonia,  nephritis,  phosphorus  pois- 
oning, grave  anaemias,  fasting  and  a  deranged  digestive  function; 
it  also  frequently  accompanies  auto-intoxication  and  chloroform 
and  ether  anaesthesia.  The  types  of  acetonuria  most  frequently  met 
with  are  those  noted  in  febrile  conditions  and  in  advanced  cases 
of  diabetes  mellitus. 

Experiments. 

1.  Isolation  from  the  Urine. — In  order  to  facilitate  the  detection 
of  acetone  in  the  urine,  the  specimen  under  examination  should  be 
distilled  and  the  tests  as  given  below  applied  to  the  resulting  dis- 
tillate. If  it  is  not  convenient  to  distil  the  urine,  the  tests  may 
be  conducted  upon  the  undistilled  fluid.  To  obtain  an  acetone  dis- 
tillate proceed  as  follows :  Place  100-250  c.c.  of  urine  in  a  distilla- 
tion flask  or  retort  and  render  it  acid  with  acetic  acid.  Collect  about 
one-third  of  the  original  volume  of  fluid  as  a  distillate,  add 
5  drops  of  10  per  cent  hydrochloric  acid  and  redistil  about  one- 
half  of  this  volume.  With  this  final  distillate  conduct  the  tests  as 
given  below. 

2.  Gunning's  Iodoform  Test. — To  about  5  c.c.  of  the  urine  or 
distillate  in  a  test-tube  add  a  few  drops  of  Lugol's  solution1  or 
ordinary  iodine  solution  (I  in  KI)  and  enough  NH4OH  to  form 
a  black  precipitate  (nitrogen  iodide).  Allow  the  tube  to  stand 
(the  length  of  time  depending  upon  the  content  of  acetone  in 
the  fluid  under  examination)  and  note  the  formation  of  a  yellow- 
ish sediment  consisting  of  iodoform.  Examine  the  sediment  under 
the  microscope  and  compare  the  form  of  the  crystals  with  those 
shown  in  Fig.  6,  p.  42.  If  the  crystals  are  not  well  formed  recrys- 
tallize  them  from  ether  and  examine  again.  The  crystals  of  iodo- 
form should  not  be  confounded  with  those  of  stellar  phosphate  (Fig. 
76,  p.  224)  which  may  be  formed  in  this  test,  particularly  if  made 
upon  the  undistilled  urine.  This  test  is  preferable  to  Lieben's  test 
(4)  since  no  substance  other  than  acetone  will  produce  iodoform 
when  treated  according  to  the  directions  for  this  test ;  both  alcohol 
and  aldehyde  yield  iodoform  when  tested  by  Lieben's  test. 

1  Lugol's  solution  may  be  prepared  by  dissolving  4  grams  of  iodine  and  6  grams 
of  potassium  iodide  in  100  c.c.  of  distilled  water. 


URINE. 

Gunning's  test  is  rather  the  most  satisfactory  test  yet  suggested 
for  the  detection  of  acetone,  and  may  he  used  with  good  results 
even  upon  the  undistilled  urine.  In  some  instances  where  the 
amount  of  acetone  present  is  very  small  it  is  necessary  to  allow 
the  tube  to  stand  24  hours  he  fore  making  the  examination  for 
iodoform  crystals.  This  test  serves  to  detect  acetone  when  present 
in  the  ratio    1  :  100,000. 

3.  Legal's  Test. — Introduce  about  5  c.c.  of  the  urine  or  distil- 
late into  a  test-tube,  add  a  few  drops  of  a  freshly  prepared  aqueous 
solution  of  sodium  nitroprusside  and  render  the  mixture  alkaline 
with  potassium  hydroxide.  A  ruby  red  color,  due  to  creatinine,  a 
normal  urinary  constituent,  is  produced  (see  WeyTs  test,  p.  278). 
Add  an  excess  of  acetic  acid  and  if  acetone  is  present  the  red  color 
will  be  intensified,  whereas  in  the  absence  of  acetone  a  yellow  color 
will  result.  Make  a  control  test  upon  normal  urine  to  show  that 
this  is  so.  A  similar  red  color  may  be  produced  by  paracreso]  in 
urines  containing  no  acetone. 

4.  Lieben's  Test. — Introduce  5  c.c.  of  the  urine  or  distillate 
into  a  test-tube,  render  it  alkaline  with  potassium  hydroxide  and 
add  t-2  c.c.  of  iodine  solution,  drop  by  drop.  If  acetone  is  present 
a  yellowish  precipitate  of  iodoform  will  be  produced.  Identify 
the  iodoform  by  means  of  its  characteristic  odor  and  its  typical 
crystalline  form  (see  Fig.  6,  p.  42).  While  fully  as  delicate  as  Gun- 
ning's test  (2)  this  test  is  not  as  accurate  since  by  means  of  the 
procedure  involved,  either  alcohol  or  aldehyde  will  yield  a  precipi- 
tate of  iodoform.  This  test  is  especially  liable  to  lead  to  erroneous 
deductions  when  urines  from  the  advanced  stages  of  diabetes  are 
under  examination,  because  of  the  presence  of  alcohol  formed  from 
the  sugar  through  fermentative  processes.1 

5.  Reynolds-Gunning  Test. — This  test  depends  upon  the  solu- 
bility of  mercuric  oxide  in  acetone  and  is  performed  as  follows: 
To  5  c.c.  of  the  urine  or  distillate  add  a  few  drops  of  mercuric 
chloride,  render  the  solution  alkaline  with  potassium  hydroxide  and 
add  an  equal  volume  of  95  per  cent  alcohol.  Shake  thoroughly  in 
order  to  bring  the  major  portion  of  the  mercuric  oxide  into  so- 
lution and  filter.  Render  the  clear  filtrate  faintly  acid  with  hy- 
drochloric acid  and  stratify  some  ammonium  sulphide,   (Nil       - 

1  Welker  reports  the  production  of  a  pink  or  red  color  during  the  application 
of  this  test  to  the  distillates  from  pathological  urines  which  had  been  preserved 
with  powdered  thymol.  He  found  the  color  to  be  due  to  an  iodothymol  com- 
pound   which    had    been    previously    prepared    synthetically    by    Messinger    and 

Vortmann. 


330  PHYSIOLOGICAL    CHEMISTRY. 

upon  this  acid  solution.  At  the  zone  of  contact  a  blackish-gray 
ring-  of  precipitated  mercuric  sulphide,  HgS,  will  form.  Alde- 
hyde also  responds  to  this  test.  Aldehyde,  however,  has  never 
been  detected  in  the  urine  and  could  only  be  present  in  this  instance 
if  the  acidified  urine  was  distilled  too  far. 

6.  Taylor's  Test. — To  10  c.c.  of  the  urine  or  distillate  in  a  test- 
tube  add  a  few  drops  of  a  freshly  prepared  aqueous  solution  of 
sodium  nitroprusside  and  stratify  concentrated  ammonium  hydrox- 
ide upon  the  mixture.  The  production  of  a  magenta  color  at  the 
point  of  contact  indicates  the  presence  of  acetone  in  the  urine  or 
distillate  under  examination.  Normal  urine  yields  an  orange-red 
color  when  subjected  to  this  technique. 

CH3 

DIACETIC  ACID,  C  =  0 

CH2-COOH. 

Diacetic  or  acetoacetic  acid  occurs  in  the  urine  only  under  patho- 
logical conditions  and  is  rarely  found  except  associated  with  acetone. 
It  is  formed  from  /?-oxybutyric  acid,  another  of  the  acetone  bodies, 
and  upon  decomposition  yields  acetone  and  carbon  dioxide.  Dia- 
ceturia  occurs  ordinarily  under  the  same  conditions  as  the  patholog- 
ical acetonuria,  i.  e.,  in  fevers,  diabetes,,  etc.  (see  p.  328).  If  very 
little  diacetic  acid  is  formed  it  may  all  be  transformed  into  acetone, 
whereas  if  a  larger  quantity  is  produced  both  acetone  and  diacetic 
acid  may  be  present  in  the  urine.  Diaceturia  is  most  frequently 
observed  in  children,  especially  accompanying  fevers  and  digestive 
disorders ;  it  is  perhaps  less  frequently  observed  in  adults,  but  when 
present,  particularly  in  fevers  and  diabetes,  it  is  frequently  followed 
by  fatal  coma. 

Diacetic  acid  is  a  colorless  liquid  which  is  miscible  with  water, 
alcohol,  and  ether,  in  all  proportions.  It  differs  from  acetone  in 
giving  a  violet-red  or  Bordeaux-red  color  with  a  dilute  solution  of 
ferric  chloride. 

Experiments. 

1.  Gerhardt's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  ferric 
chloride  solution,  drop  by  drop,  until  no  more  precipitate  forms. 
In  the  presence  of  diacetic  acid  a  Bordeaux-red  color  is  produced ; 
this  color  may  be  somewhat  masked  by  the  precipitate  of  ferric 
phosphate,  in  which  case  the  fluid  should  be  filtered. 


UK1NE.  331 

A  positive  result  from  the  above  manipulation  simply  indicates 
the  possible  presence  of  diacetic  acid.  Before  making  a  final  de- 
cision regarding  the  presence  of  this  body  make  the  two  Following 

control  experiments  : 

(a)  Place  5  c.c.  of  urine  in  a  test-tube  and  boil  it  vigorously  for 
3-5  minutes.  Cool  the  tube  and,  with  the  boiled  urine,  make  the  tesl 
as  given  on  p.  330.  As  has  been  already  stated,  diacetic  acid  yields 
acetone  upon  decomposition  and  acetone  duo  not  give  a  Bordeaux- 
red  color  with  ferric  chloride.  By  boiling  as  indicated  above,  there- 
fore, any  diacetic  acid  present  would  be  decomposed  into  acetone 
and  carbon  dioxide  and  the  test  upon  the  resulting  fluid  would  be 
negative.  If  positive  the  color  is  due  to  the  presence  .of  bodies 
other  than  diacetic  acid. 

(b)  Place  5  c.c.  of  urine  in  a  test-tube,  acidify  with  H2S04, 
to  free  diacetic  acid  from  its  salts,  and  carefully  extract  the  mix- 
ture with  ether  by  shaking.  If  diacetic  acid  is  present  it  will  be 
extracted  by  the  ether.  Now  remove  the  ethereal  solution  and 
add  to  it  an  equal  volume  of  dilute  ferric  chloride ;  diacetic  acid  is 
indicated  by  the  production  of  the  characteristic  Bordeaux-red  color. 
This  color  disappears  spontaneously  in  24-48  hours.  Such  sub- 
stances as  antipyrin,  kairin,  phenacetin,  salicylic  acid,  salicylates, 
sodium  acetate,  thiocyanates  and  thallin  yield  a  similar  red  color 
under  these  conditions,  but  when  due  to  the  presence  of  any  of 
these  substances  the  color  does  not  disappear  spontaneously  but 
may  remain  permanent  for  days.  Many  of  these  disturbing  sub- 
stances are  soluble  in  benzene  or  chloroform  and  may  be  removed 
from  the  urine  by  this  means  before  extracting  with  ether  as  above. 
Diacetic  acid  is  insoluble  in  benzene  or  chloroform. 

2.  Arnold-Lipliawsky  Reaction. — This  reaction  is  somewhat 
more  delicate  than  Gerhardt's  test  ( 1 )  and  serves  to  detect  diacetic 
acid  when  present  in  the  proportion  of  1  :  25,000.  It  is  also  negative 
toward  acetone,  /3-oxybutyric  acid  and  the  interfering  drugs  men- 
tioned as  causing  erroneous  deductions  in  the  application  of  Ger- 
hardt's test.  If  the  urine  under  examination  is  highly  pigmented 
it  should  be  partly  decolorized  by  means  of  animal  charcoal  before 
applying  the  test  as  indicated  below. 

Place  5  c.c.  of  the  urine  under  examination  and  an  equal  volume 
of  the  Arnold-Lipliawsky  reagent1  in  a  test  tube,  add  a  few  drops 

1  This  reagent  consists  of  two  definite  solutions  which  are  ordinarily  preserved 
separately  and  mixed  just  before  using.  The  two  solutions  are  prepared  as 
follows  : 

(a)   One  per  cent  aqueous  solution  of  potassium  nitrite. 


332  PHYSIOLOGICAL    CHEMISTRY. 

of  concentrated  ammonia  and  shake  the  tube  vigorously.  Note 
the  production  of  a  brick-red  color.  Take  1-2  c.c.  of  this  colored 
solution,  add  10-20  c.c.  of  hydrochloric  acid  (sp.  gr.  1.19),  3  c.c. 
of  chloroform  and  2-4  drops  of  ferric  chloride  solution  and  care- 
fully mix  the  fluids  without  shaking.  Diacetic  acid  is  indicated  by 
the  chloroform  assuming  a  violet  or  blue  color ;  if  diacetic  acid  is 
absent  the  color  may  be  yellow  or  light  red. 

H       OH  H 

/3-OXYBUTYRIC  ACID,      H  — C  —  C  —  C  —  COOH. 

H      H     H 

This  acid  does  not  occur  as  a  normal  constituent  of  urine  but  is 
found  only  under  pathological  conditions  and  then  always  in  eon- 
junction  with  either  acetone  or  diacetic  acid.  Either  of  these  bodies 
may  be  formed  from  /3-oxybutyric  acid  under  proper  conditions.  It 
is  present  in  especially  large  amount  in  severe  cases  of  diabetes  and 
has  also  been  detected  in  digestive  disturbances,  continued  fevers, 
scurvy,  measles  and  in  starvation.  It  is  probable  that,  in  man,  /?- 
oxybutyric  acid,  in  common  with  acetone  and  diacetic  acid,  arises 
principally  from  the  breaking  down  of  fatty  tissues  within  the  or- 
ganism. The  condition  in  which  large  amounts  of  acetone  and 
diacetic  acid,  and  in  severe  cases  /?-oxybutyric  acid  also,  are  excreted 
in  the  urine  is  known  as  "  acidosis."  In  diabetes  the  deranged 
metabolic  conditions  cause  the  production  of  great  quantities  of 
these  substances  which  lead  to  an  acid  intoxication  and  ultimately 
to  diabetic  coma. 

Ordinarily  /3-oxybutyric  acid  is  an  odorless,  transparent  syrup, 
which  is  lsevorotatory  and  easily  soluble  in  water,  alcohol  and  ether ; 
it  may  be  obtained  in  crystalline  form. 

Experiments. 

1.  Black's  Reaction. — Inasmuch  as  the  urinary  pigments  as  well 
as  any  contained  sugar  or  diacetic  acid  will  interfere  with  the  deli- 
cacy of  this  test  when  applied  to  the  urine  directly  the  following 
preliminary  procedure   is  necessary :     Concentrate    10  c.c.   of  the 

(b)  One  gram  of  />-amino-acetophenon  dissolved  in  100  c.c.  of  distilled  water 
and  enough  hydrochloric  acid  (about  2  c.c.)  added,  drop  by  drop,  to  cause  the 
solution,  which  is  at  first  yellow,  to  become  entirely  colorless.  An  excess  of 
acid  must  be  avoided. 

Before  using,  a  and  b  are  mixed  in  the  ratio  1  :  2. 


urine.  333 

urine  under  examination  to  one-third  or  one  fourth  of  its  original 
volume  in  an  evaporating  dish  at  a  gentle  heat.  Acidif)  the  resi 
due  with  a  few  drops  of  concentrated  hydrochloric  arid,  add  suffi- 
cient plaster  of  Paris  to  make  a  thick  paste  and  allow  the  mixture 
to  stand  until  it  begins  to  "set."  It  should  now  he  stirred  and 
broken  up  in  the  dish  by  means  of  a  stirritlg  rod  with  a  blunl  end. 
Extract  the  porous  meal  thus  produced  twice  with  ether  by  stirring 
and  decantation.  Any  /?-oxybutyric  acid  present  will  be  extracted 
by  the  ether.  Evaporate  the  ether  extract  spontaneously  or  on  a 
water-bath,  dissolve  the  residue  in  water  and  neutralize  it  with 
barium  carbonate.  To  five  to  ten  c.c.  of  this  neutral  fluid  in  a 
tube  add  two  to  three  drops  of  ordinary  commercial  acid  hydro-en 
peroxide.  Mix  by  shaking  and  add  a  few  drops  of  Black's  reagent.1 
Permit  the  tube  to  stand  and  note  the  gradual  development  of  a 
rose  color  which  increases  to  its  maximum  intensity  and  then 
gradually  fades.2 

In  carrying  out  the  test  care  should  be  taken  to  see  that  the 
solution  is  cold  and  approximately  neutral  and  that  a  large  excess 
of  hydrogen  peroxide  and  Black's  reagent  are  not  added.  In  case 
but  little  /?-oxybutyric  acid  is  present  the  color  will  fail  to  appear  or 
will  be  but  transitory  if  the  oxidizing  agents  are  added  in  too  great 
excess.  It  is  preferable  to  add  a  few  drops  of  the  reagent  and  at 
intervals  of  a  few  minutes  repeat  the  process  until  the  color  under- 
goes no  further  increase  in  intensity.  One  part  of  /8-oxybutyric 
acid  in  10,000  parts  of  the  solution  may  be  detected  by  this  test. 

2.  Polariscopic  Examination. — Subject  some  of  the  urine  (free 
from  protein)  to  the  ordinary  fermentation  test  (see  page  313). 
This  will  remove  dextrose  and  laevulose,  which  would  interfere  with 
the  polariscopic  test.  Now  examine  the  fermented  fluid  in  the 
polariscope  and  if  it  is  l?evorotatory  the  presence  of  /3-oxybutyric 
acid  is  indicated.  This  test  is  not  absolutely  reliable,  however, 
since  conjugate  glycuronates  are  also  l?evorotatory  after  fermenta- 
tion. 

3.  Kiilz's  Test. — Evaporate  the  urine,  after  fermenting  it  as 
indicated  in  the  last  test,  to  a  syrup,  add  an  equal  volume  of  con- 
centrated sulphuric  acid  and  distil  the  mixture  directly  without  cool- 
ing. Under  these  conditions  a-crotonic  acid  is  formed  and  is 
. present  in  the  distillate.     Allow  the  distillate  to  cool   slowly  and 

'Made  by  dissolving  five  grams  of  ferric  chloride  ami  0.4  gram  of  ferrous 
chloride  in  100  c.c.  of  water. 

"This  disappearance  of  color  is  due  to  the  further  oxidation  of  the  diacetic 
acid. 


334  PHYSIOLOGICAL    CHEMISTRY. 

note  the  formation  of  crystals  of  a-crotonic  acid  which  are  soluble 
in  ether  ancl  melt  at  72  °  C.  In  case  very  slight  traces  of  j8-oxy- 
butyric  acid  be  present  in  the  urine  under  examination  the  amount 
of  a-crotonic  acid  formed  may  be  too  small  to  yield  a  crystalline 
product.  In  this  event  the  distillate  should  be  extracted  with  ether, 
the  ethereal  extract  evaporated  and  the  residue  washed  with  water. 
Under  these  conditions  the  impurities  will  be  removed  and  the  a- 
crotonic  acid  will  remain  behind  as  a  residue.  The  melting-point 
of  this  residue  may  then  be  determined. 

CONJUGATE  GLYCURONATES. 

Glycuronic  acid  does  not  occur  free  in  the  urine  but  is  found,  for 
the  most  part,  in  combination  with  phenol.  Much  smaller  quan- 
tities are  excreted  in  combination  with  indoxyl  and  skatoxyl.  The 
total  content  of  conjugate  glycuronates  seldom  exceeds  0.004  Per 
cent  under  normal  conditions.  The  output  may  be  very  greatly  in- 
creased as  the  result  of  the  administration  of  antipyrin,  borneol, 
camphor,  chloral,  menthol,  morphine,  naphthol,  turpentine,  etc. 
The  glycuronates  as  a  group  are  lsevorotatory,  whereas  glycuronic 
acid  is  dextrorotatory.  Most  of  the  glycuronates  reduce  alkaline 
metallic  oxides  and  so  introduce  an  error  in  the  examination  of 
urine  for  sugar.  Conjugate  glycuronates  often  occur  associated 
with  dextrose  in  glycosuria,  diabetes  mellitus  and  in  some  other 
disorders.     As  a  class  the  glycuronates  are  non-fermentable. 

Experiments. 

1.  Fermentation-Reduction  Test. — Test  the  urine  by  Fehling's 
test.  If  there  is  reduction  try  Barfoed's  test.  If  negative  this 
indicates  the  absence  of  monosaccharides.  A  negative  fermentation 
test  would  now  indicate  the  presence  of  conjugate  glycuronates 
(or  lactose  in  rare  cases).1 

If  dextrose  is  present  in  the  urine  tested  for  glycuronates  the 
urine  must  first  be  subjected  to  a  polariscopic  examination,  then 
fermented  and  a  second  polariscopic  examination  made.  The  sugar 
being  dextrorotatory  and  fermentable  and  the  glycuronates  being 
lsevorotatory  and  non-fermentable  the  second  polariscopic  test  will 
show  a  lsevorotation  indicative  of  conjugate  glycuronates. 

2.  Tollens'  Reaction. — Make  this  test  according  to  directions 
given  under  Pentoses,  page  37. 

3  If  necessary  to  differentiate  between  lactose  and  glycuronates  apply  the  mucic 
acid  test  (see  p.  337)  or  the  phenylhydrazine  reaction  (see  p.  306). 


urine.  335 

PENTOSES. 

We  have  two  distinct  types  of  pentosuria,  /'.  <?.,  alimentary  pen- 
tosuria, resulting  from  the  ingestion  of  large  quantities  of  pentose- 
rich  vegetables  such  as  prunes,  cherries,  grapes  or  plums,  and 
fruit  juices,  in  which  condition  the  pentoses  appear  only  temporarily 
in  the  urine;  and  the  chronic  form  of  pentosuria,  in  which  the  output 
of  pentoses  bears  no  relation  whatever  to  the  quantity  and  nature  of 
the  pentose  content  of  the  food  eaten.  In  occurring  in  these 
two  forms,  pentosuria  resembles  glycosuria  (see  page  306),  but  it  is 
definitely  known  that  pentosuria  bears  no  relation  to  diabetes  niel- 
litus  and  there  is  no  generally  accepted  theory  to  account  for  the 
occurrence  of  the  chronic  form  of  pentosuria.  The  pentose  de- 
tected most  frequently  in  the  urine  is  arabinose,  the  inactive  form 
generally  occurring  in  chronic  pentosuria  and  the  kevorotatory 
variety  occurring  in  the  alimentary  type  of  the  disorder. 

Experiments. 

1.  Tollens'  Reaction. — To  equal  volumes  of  urine  and  hydro- 
chloric acid  (sp.  gr.  1.09)  add  a  little  phloroglucin  and  heat  the 
mixture  on  a  boiling  water-bath.  Pentose,  galactose,  or  glycuronic 
acid  will  be  indicated  by  the  appearance  of  a  red  color.  To  differen- 
tiate between  these  bodies  examine  by  the  spectroscope  and  look 
for  the  absorption  band  between  D  and  E  given  by  pentoses  and 
glycuronic  acid,  and  then  differentiate  between  the  two  latter  bodies 
by  the  melting-points  of  their  osazones. 

2.  Orcin  Test. — Place  equal  volumes  of  urine  and  hydrochloric 
acid  (sp.  gr.  1.09)  in  a  test-tube,  add  a  small  amount  of  orcin, 
and  heat  the  mixture  to  boiling-.  Color  changes  from  red,  through 
reddish-blue  to  green  will  be  noted.  When  the  solution  becomes 
green  it  should  be  shaken  in  a  separatory  funnel  with  a  little  amy! 
alcohol,  and  the  alcoholic  extract  examined  spectroscopically.  An 
absorption  band  between  C  and  D  will  be  observed. 


FAT. 

When  fat  finds  its  way  into  the  urine  through  a  lesion  which 
brings  some  portion  of  the  urinary  passages  into  communication 
with  the  lymphatic  system  a  condition  known  as  chyluria  is  es- 
tablished. The  turbid  or  milky  appearance  of  such  urine  is  due 
to  its  content  of  chvle.     This  disease  is  encountered  mosl  frequently 


336  PHYSIOLOGICAL    CHEMISTRY. 

in  tropical  countries,  but  is  not  entirely  unknown  in  more  temperate 
climates.  Albumin  is  a  constant  constituent  of  the  urine  in  chyluria. 
Upon  shaking  a  chylous  urine  with  ether  the  fat  is  dissolved  by  the 
ether  and  the  urine  becomes  clearer  or  entirely  clear. 


H^MATOPORPHYRIN. 

Urine  containing  this  body  is  occasionally  met  with  in  various 
diseases  but  more  frequently  after  the  use  of  quinine,  tetronal,  tri- 
onal  and  especially  sulphonal.  Such  urines  ordinarily  possess  a 
reddish  tint,  the  depth  of  color  varying  greatly  under  different  con- 
ditions. 

Experiments. 

1.  Spectroscopic  Examination. — To  100  c.c.  of  urine  add  about 
20  c.c.  of  a  10  per  cent  solution  of  potassium  hydroxide  or  ammon- 
ium hydroxide.  The  precipitate  which  forms  consists  principally  of 
earthy  phosphates  to  which  the  haematoporphyrin  adheres  and  is 
carried  down.  Filter  off  the  precipitate,  wash  it  and  transfer  to 
a  flask  and  warm  with  alcohol  acidified  with  hydrochloric  acid. 
By  this  process  the  haematoporphyrin  is  dissolved  and  on  filtering 
will  be  found  in  the  filtrate  and  may  be  identified  by  means  of  the 
spectroscope  (see  page  207,  and  Absorption  Spectra,  Plate  II). 

2.  Acetic  Acid  Test. — To  100  c.c.  of  urine  add  5  c.c.  of  glacial 
acetic  acid  and  allow  the  mixture  to  stand  48  hours.  Haematopor- 
phyrin  deposits  in  the  form  of  a  precipitate. 


LACTOSE. 

Lactose  is  rarely  found  in  the  urine  except  as  it  is  excreted  by 
women  during  pregnancy,  during  the  nursing  period  or  soon  after 
weaning.  It  is  rather  difficult  to  show  the  presence  of  lactose  in 
the  urine  in  a  satisfactory  manner,  since  the  formation  of  the 
characteristic  lactosazone  is  not  attended  with  any  great  measure 
of  success  under  these  conditions.  It  is,  however,  comparatively 
easy  to  show  that  it  is  not  dextrose,  for,  while  it  responds  to  re- 
duction tests,  it  does  not  ferment  with  pure  yeast  and  does  not 
give  a  dextrosazone.  An  absolutely  conclusive  test,  of  course,  is 
the  isolation  of  the  lactose  in  crystalline  form  (Fig.  75,  p.  220) 
from  the  urine. 

On  oxidation  with  nitric  acid  lactose  and  galactose  yield  mucic 


urine.  337 

acid.  This  test  is  frequently  used  in  urine  examination  to  differen- 
tiate lactose  and  galactose  from  other  reducing  sugars. 

Experiments. 

i.  Mucic  Acid  Test. — Treat  ioo  c.c.  of  the  urine  under  examina- 
tion with  20  c.c.1  of  concentrated  nitric  acid  and  evaporate  the  mix- 
ture in  a  broad,  shallow  glass  vessel,  upon  a  boiling  water-bath 

until  the  volume  of  the  solution  is  only  about  20  c.c.  At  this 
point  the  fluid  should  be  clear  and  a  fine  white  precipitate  of  mucic 
acid  should  separate.  If  the  percentage  of  lactose  in  the  urine 
is  low  it  may  be  necessary  to  cool  the  solution  and  permit  it  to  stand 
for  some  time  before  the  precipitate  will  form.  It  is  impossible 
to  differentiate  between  galactose  and  lactose  by  means  of  this 
test,  but  the  reaction  does  serve  to  differentiate  these  two 
sugars  from  all  other  reducing  sugars.  A  satisfactory  differentia- 
tion between  lactose  and  galactose  may  be  made  by  means  of 
Barfoed's  test,  p.  313. 

2.  Rubner's  Test. — To  10  c.c.  of  urine  in  a  small  beaker  add 
some  plumbic  acetate,  in  substance,  heat  to  boiling  and  add  NH4OH 
until  no  more  precipitate  is  dissolved.  In  the  presence  of  lactose 
a  brick-red  or  rose-red  color  develops,  whereas  dextrose  gives  a 
coffee-brown  color,  maltose  a  light  yellow  color  and  kevulose  no 
color  at  all  under  the  same  conditions. 

3.  Compound  Test. — Try  the  phenylhydrazine  test,  the  fermen- 
tation test  and  Barfoed's  test  according  to  directions  given  under 
Dextrose,  pages  306,  313  and  314.  If  these  are  negative,  try 
Nylander's  test,  page  312.  If  this  last  test  is  positive,  the  presence 
of  lactose  is  indicated. 

GALACTOSE. 

Galactose  has  occasionally  been  detected  in  the  urine,  and  in  par- 
ticular in  that  of  nursing  infants  afflicted  with  a  deranged  digestive 
function.  Lactose  and  galactose  may  be  differentiated  from  other 
reducing  sugars  which  may  be  present  in  the  urine  by  means  vi  the 
mucic  acid  test.  This  test  simply  consists  in  the  production  of 
mucic  acid  through  oxidation  of  the  sugar  with  nitric  acid. 

1  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  i<  necessary  t"  use  25-35 
c.c.  of  nitric  acid.  Under  these  conditions  the  mixture  should  be  evaporated 
until  the  remaining  volume  is  approximately  equivalent  to  that  of  the  nitric 
acid  added. 


338  physiological  chemistry. 

Experiments. 

i.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  urine  under  examin- 
ation with  20  c.c.1  of  concentrated  nitric  acid  and  evaporate  the 
mixture  in  a  broad,  shallow  glass  vessel,  upon  a  boiling  water- 
bath,  until  the  volume  of  the  solution  is  only  about  20  c.c.  At 
this  point  the  fluid  should  be  clear  and  a  fine,  white  precipitate 
of  mucic  acid  should  separate.  If  the  percentage  of  galactose  pres- 
ent in  the  urine  is  low  it  may  be  necessary  to  cool  the  solution  and 
permit  it  to  stand  for  some  time  before  the  precipitate  will  form. 
It  is  impossible  to  differentiate  between  galactose  and  lactose  by 
means  of  this  test,  but  the  reaction  does  serve  to  differentiate  these 
two  sugars  from  all  other  reducing  sugars.  A  satisfactory  differ- 
entiation between  galactose  and  lactose  may  be  made  by  Barfoed's 
test,  p.  313. 

2.  Tollens'  Reaction. — To  equal  volumes  of  the  urine  and  hy- 
drochloric acid  (sp.  gr.  1.09)  add  a  little  phloroglucin  and  heat  the 
mixture  on  a  boiling  water-bath.  Galactose,  pentose  and  glycur- 
onic  acid  will  be  indicated  by  the  appearance  of  a  red  color.  Galac- 
tose may  be  differentiated  from  the  two  latter  substances  in  that 
its  solutions  exhibit  no  absorption  bands  upon  spectroscopical  ex- 
amination. 

LiEVULOSE. 

Diabetic  urine  frequently  possesses  the  power  of  rotating  the 
plane  of  polarized  light  to  the  left,  thus  indicating  the  presence  of 
a  lsevorotatory  substance.  This  lsevorotation  is  sometimes  due  to 
the  presence  of  lsevulose,  although  not  necessarily  confined  to  this 
carbohydrate,  since  conjugate  glycuronates  and  /?-oxybutyric  acid, 
two  other  lsevorotatory  bodies,  are  frequently  found  in  the  urine  of 
diabetics.  Lsevulose  is  invariably  accompanied  by  dextrose  in  dia- 
betic urine,  but  Iccvulosuria  has  been  observed  as  a  separate  anomaly. 
The  presence  of  lsevulose  may  be  inferred  when  the  percentage 
of  sugar,  as  determined  by  the  titration  method,  is  greater  than  the 
percentage  indicated  by  the  polariscopic  examination. 

Experiments. 

1.  Borchardt's  Reaction. — To  about  5  c.c.  of  urine  in  a  test- 
tube  add  an  equal  volume  of  25  per  cent  hydrochloric  acid  and  a 
few  crystals  of  resorcin.     Heat  to  boiling  and  after  the  production 

1  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35 
c.c.  of  nitric  acid.  Under  these  conditions  the  mixture  should  be  evaporated 
until  the  remaining  volume  is  approximately  equivalent  to  that  of  the  nitric 
acid  added. 


urine.  339 

of  a  red  color,  cool  the  tube  under  running  writer  and  transfer  to 
an  evaporating-  dish  or  beaker.  Make  the  mixture  slightly  alka- 
line with  solid  potassium  hydroxide,  return  il  to  a  tesl  tube,  add 
2-3  c.c.  of  acetic  ether  and  -hake  the  tube  vigorously.  In  the 
presence  of  laevulose  the  acetic  ether  is  colored   yell 

The  only  urinary  constituents  which  interfere  with  the  test  are 
nitrites  and  indican  and  these  interfere  only  when  they  are  simul- 
taneously present.  Under  these  conditions,  the  urine  should  be 
acidified  with  acetic  acid  and  heated  to  boiling  for  one  minute  to  re- 
move the  nitrites.  In  case  the  indican  content  is  very  large,  it  will 
impart  a  blue  color  to  the  acetic  ether,  thus  masking-  the  yellow  color 
due  to  laevulose.  When  such  urines  are  to  he  examined,  the  indi- 
can should  first  be  removed  by  Obermayer's  test  (see  p.  281).  The 
chloroform  should  then  be  discarded,  the  acid-urine  mixture  diluted 
with  one-third  its  volume  of  water  and  the  test  applied  as  described 
above.  The  urine  of  patients  who  have  ingested  santonin  or  rhu- 
barb, respond  to  the  test.  The  test  will  serve  to  detect  laevulose 
when  present  in  a  dilution  of  1  :  2000  i.  e.,  0.05  per  cent. 

2.  Seliwanoff's  Reaction. — To  5  c.c.  of  Seliwanoff's  reagent1 
in  a  test-tube  add  a  few  drops  of  the  urine  under  examination  and 
heat  the  mixture  to  boiling.  The  presence  of  laevulose  is  indicated 
by  the  production  of  a  red  color  and  the  separation  of  a  red  precipi- 
tate. The  latter  may  be  dissolved  in  alcohol  to  which  it  will  impart 
a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained 
with  urines  containing   dextrose. 

3.  Phenylhydrazine  Test. — Make  the  test  according  to  direc- 
tions under  Dextrose,  1,  page  306. 

4.  Polariscopic  Examination. — A  simple  polariscopic  examina- 
tion, when  taken  in  connection  with  other  ordinary  tests,  will  fur- 
nish the  requisite  data  regarding  the  presence  of  laevulose,  provided 
laevulose  is  not  accompanied  by  other  laevorotatory  substances,  such 
as  conjugate  glycuronates  and  j8-oxybutyric  acid. 

CHOH 

//\ 
HOHC      CHOH 

INOSITE, 

HOHC      CHOH 
\/ 

nroH 

1  Seliwanoff's  reagent  may  be  preparol  by  dissolving  0.05  gram  of  resorcin  in 

100  c.c.  of  dilute  (1:2)   hydrochloric  acid. 


34-0  PHYSIOLOGICAL    CHEMISTRY. 

Inosite  occasionally  occurs  in  the  urine  in  albuminuria,  diabetes 
mellitus  and  diabetes  insipidus.  It  is  claimed  also  that  copious 
water-drinking  causes  this  substance  to  appear  in  the  urine.  Inosite 
was  at  one  time  considered  to  be  a  sugar  but  is  now  known  to  be 
hexahydroxybenzene,  as  the  formula  on  p.  339  indicates.  It  is  an 
example  of  a  non-carbohydrate  in  whose  molecule  the  H  and  O 
are  present  in  the  proportion  to  form  water.  In  other  words  it  has 
the  formula  of  the  hexoses,  i.  e.,  C6H1206.  Inosite  occurs  widely 
distributed  in  the  vegetable  kingdom  and  because  of  this  fact  the 
theory  has  been  voiced  that  it  represents  one  of  the  first  stages  in  the 
conversion  of  a  carbohydrate  into  the  benzene  ring.  It  is  found  in 
the  liver,  spleen,  lungs,  brain,  kidneys,  suprarenal  capsules,  muscles, 
leucocytes,  testes  and  urine  under  normal  conditions. 

Experiment. 

1.  Detection  of  Inosite. — Acidify  the  urine  with  concentrated 
nitric  acid  and  evaporate  nearly  to  dryness.  Add  a  few  drops  of 
ammonium  hydroxide  and  a  little  calcium  chloride  solution  to  the 
moist  residue  and  evaporate  the  mixture  to  dryness.  In  the  pres- 
ence of  inosite  (0.001  gram)   a  bright  red  color  is  obtained. 


LAIOSE. 

This  substance  is  occasionally  found  in  the  urine  in  severe  cases 
of  diabetes  mellitus.  By  some  investigators  laiose  is  classed  with 
the  sugars.  It  resembles  lsevulose  in  that  it  has  the  property  of 
reducing  certain  metallic  oxides  and  is  lsevorotatory,  but  differs 
from  lgevulose  in  being  amorphous,  non-fermentable  and  in  not  pos- 
sessing a  sweet  taste. 

MELANINS. 

These  pigments  never  occur  normally  in  the  urine  but  are  present 
under  certain  pathological  conditions,  their  presence  being  especially 
associated  with  melanotic  tumors.  Ordinarily  the  freshly  passed 
urine  is  clear,  but  upon  exposure  to  the  air  the  color  deepens  and 
may  at  the  last  be  very  dark  brown  or  black  in  color.  The  pig- 
ment is  probably  present  in  the  form  of  a  chromogen  or  melanogen 
and  upon  coming  in  contact  with  the  air  oxidation  occurs,  causing 
the  transformation  of  the  melanogen  into  melanin  and  consequently 
the  darkening  of  the  urine. 


URINE.  .541 

It  is  claimed  that  melanuria  is  proof  of  the  formation  of  a  vis- 
ceral melanotic  growth.  In  many  instance-,  without  doubt,  urines 
rich  in  indican  have  been  wrongly  taken  as  diagnostic  proof  of 
melanuria.  The  pigment  melanin  is  sometimes  mistaken  for  indigo 
and  melanogen  for  indican.  It  is  comparatively  easy  to  differentiate 
between  indigo  and  melanin  through  the  solubility  of  the  former  in 
chloroform. 

In  rare  cases  melanin  is  found  in  urinary  sediment  in  the  form 
of  fine  amorphous  granules. 

Experiments. 

1.  Zeller's  Test. — To  50  c.c.  of  urine  in  a  small  beaker  add  an 
equal  volume  of  bromine  water.  In  the  presence  of  melanin  a 
yellow  precipitate  will  form  and  will  gradually  darken  in  color,  ulti- 
mately  becoming   black. 

2.  von  Jaksch-Pollak  Reaction. — Add  a  few  drops  of  ferric 
chloride  solution  to  10  c.c.  of  urine  in  a  test-tube  and  note  the 
formation  of  a  gray  color.  Upon  the  further  addition  of  the  chlor- 
ide a  dark  precipitate  forms,  consisting  of  phosphates  and  adhering 
melanin.  An  excess  of  ferric  chloride  causes  the  precipitate  to 
dissolve. 

This  is  the  most  satisfactory  test  for  the  identification  of  melanin 
in  the  urine. 

UROROSEIN. 

This  is  a  pigment  which  is  not  present  in  normal  urine  but  may 
be  detected  in  the  urine  of  various  diseases,  such  as  pulmonary 
tuberculosis,  typhoid  fever,  nephritis  and  stomach  disorders.  LJro- 
rosein,  in  common  with  various  other  pigments,  does  not  occur  pre- 
formed in  the  urine,  but  is  present  in  the  form  of  a  chromogen, 
which  is  transformed  into  the  pigment  upon  treatment  with  a  min- 
eral acid. 

Experiments. 

1.  Robin's  Reaction. — Acidify  10  c.c.  of  urine  with  about  15 
drops  of  concentrated  hydrochloric  acid.  Upon  allowing  the  acidi- 
fied urine  to  stand,  a  rose-red  color  will  appear  it'  urorosein  is 
present. 

2.  Nencki  and  Sieber's  Reaction. — To  100  c.c.  of  mine  in  a 
beaker  add  10  c.c.  of  25  per  cent  sulphuric  acid.  Allow  the  acidi- 
fied urine  to  stand  and  note  the  appearance  of  a  rose-red  color. 
The  pigment  may  be  separated  by  extraction  with  amy!  alcohol. 


342  PHYSIOLOGICAL    CHEMISTRY. 

UNKNOWN    SUBSTANCES. 

Ehrlich's  Diazo  Reaction. — Place  equal  volumes  of  urine  and 
Ehrlich's  diazobenzenesulphonic  acid  reagent1  in  a  test-tube,  mix 
thoroughly  by  shaking  and  quickly  add  ammonium  hydroxide  in 
excess.  The  test  is  positive  if  both  the  fluid  and  the  foam  assume 
a  red  color.  If  the  tube  is  allowed  to  stand  a  precipitate  forms, 
the  upper  portion  of  which  exhibits  a  blue,  green,  greenish-black 
or  violet  color.  Normal  urine  gives  a  brownish-yellow  reaction 
with  the  above  manipulation. 

The  exact  nature  of  the  substance  or  substances  upon  whose 
presence  in  the  urine  this  reaction  depends  is  not  well  understood. 
Some  investigators  claim  that  a  positive  reaction  indicates  an  ab- 
normal decomposition  of  protein  material,  whereas  others  assume 
it  to  be  due  to  an  increased  excretion  of  alloxyproteic  acid,  oxy- 
proteic  acid  or  uroferric  acid. 

The  reaction  may  be  taken  as  a  metabolic  symptom  of  certain  dis- 
orders, which  is  of  value  diagnostically  only  when  taken  in  connec- 
tion with  the  other  symptoms.  The  reaction  appears  principally 
in  the  urine  in  febrile  disorders  and  in  particular  in  the  urine  in 
typhoid  fever,  tuberculosis  and  measles.  The  reaction  has  also 
been  obtained  in  the  urine  in  various  other  disorders  such  as  car- 
cinoma, chronic  rheumatism,  diphtheria,  erysipelas,  pleurisy,  pneu- 
monia, scarlet  fever,  syphilis,  typhus,  etc.  The  administration  of 
alcohol,  chrysarobin,  creosote,  cresol,  dionin,  guaiacol,  heroin,  mor- 
phine, naphthalene,  opium,  phenol,  tannic  acid,  etc.,  will  also  cause 
the  urine  to  give  a  positive  reaction. 

The  following  chemical  reactions  take  place  in  this  test: 

(a)  NaN02  +  HC1  =  HN02  +  NaCl. 

NHo  N 

/  /     \ 

(b)  C6H4  +HNOo  =  C6H4  N  +  2H20. 

\  '       .      \    / 

HS03  S03 

Sulphanilic  acid.  Diazo-benzenesulphonic  acid. 

1  Two  separate  solutions  should  be  prepared  and  mixed  in  definite  proportions 
when  needed  for  use. 

(a)   Five  grams  of  sodium  nitrite  dissolved  in  1  liter  of  distilled  water. 

(&)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid  in  1  liter  of 
distilled  water. 

Solutions  a  and  b  should  be  preserved  in  well  stoppered  vessels  and  mixed  in 
the  proportion  1  :  50  when  required.  Green  asserts  that  greater  delicacy  is  se- 
cured by  mixing  the  solutions  in  the  proportion  1  :  100.  The  sodium  nitrite  de- 
teriorates upon  standing  and  becomes  unfit  for  use  in  the  course  of  a  few  weeks. 


(MA  P 


<    X  X 


URINE:    ORGANIZED    AND    UNORGANIZED 
SEDIMENTS. 

The  data  obtained  from  carefully  conducted  microscopical  exam- 
inations of  the  sediment  of  certain  pathological  urines  arc  of  very 
great  importance,  diagnostically.  Too  little  emphasis  is  some- 
times placed  upon  the  value  of  such   findings. 


Fig.  97. 


Fig.  98. 


The    Purdv    Electric    Centrifu 


SEDIMEN1    Tl    BE    FOR    1  ill      PURDY 

Electric  Centrifuge. 


The  sedimentary  constituents  may  be  divided  into  two  classes, 
i.  e.,  organized  and  unorganized.  The  sediment  is  ordinarily  col- 
lected for  examination  by  means  of  the  centrifuge  (Fig.  07.  above). 
An  older  method,  and  one  still  in  vogue  in  some  quarters,  is  the  so- 
called  gravity  method.  This  simply  consists  in  placing  the  urine 
in  a  conical  glass  and  allowing  the  sedimenl  to  settle.  The  col- 
lection of  the  sediment  by  mean-  of  the  centrifuge,  however,  is 
much  preferable,   since  the  process  of   sedimentation   may  be  ac 

34  3 


344  PHYSIOLOGICAL    CHEMISTRY. 

complished  by  the  use  of  this  instrument  in  a  few  minutes,  and 
far  more  perfectly,  whereas  when  the  other  method  is  used  it  is 
frequently  necessary  to  allow  the  urine  to  remain  in  the  conical 
glass  12-24  hours  before  sufficient  sediment  can  be  secured  for  the 
microscopical  examination. 

(a)   Unorganized  Sediments. 

Ammonium  magnesium  phosphate  ("Triple  phosphate"). 

Calcium  oxalate.  .    . 

Calcium  carbonate. 

Calcium  phosphate. 

Calcium  sulphate. 

Uric  acid. 

Urates. 

Cystine. 

Cholesterol. 

Hippuric  acid. 

Leucine  and  tyrosine. 

Hsematoidin  and  bilirubin. 

Magnesium  phosphate. 

Indigo. 

Xanthine. 

Melanin. 

Ammonium  Magnesium  Phosphate  ("Triple  Phosphate"). 
— Crystals  of  "  triple  phosphate  "  are  a  characteristic  constituent 
of  the  sediment  when  alkaline  fermentation  of  the  urine  has  taken 
place  either  before  or  after  being  voided.  They  may  even  be  de- 
tected in  amphoteric  or  slightly  acid  urine  provided  the  ammonium 
salts  are  present  in  large  enough  quantity.  This  substance  may 
occur  in  the  sediment  in  two  forms,  i.  e.,  prisms  and  the 
feathery  type.  The  prismatic  form  of  crystal  (Fig.  96,  p.  301)  is 
the  one  most  commonly  observed  in  the  sediment ;  the  feathery 
form  (Fig.  96,  p.  301)  predominates  when  the  urine  is  made  am- 
moniacal  with  ammonia. 

The  sediment  of  the  urine  in  such  disorders  as  are  accompanied 
by  a  retention  of  urine  in  the  lower  urinary  tract  contains  "  triple 
phosphate  "  crystals  as  a  characteristic  constituent.  The  crystals 
are  frequently  abundant  in  the  sediment  during  paraplegia,  chronic 
cystitis,  enlarged  prostate  and  chronic  pyelitis. 

Calcium  Oxalate. — Calcium  oxalate  is  found  in  the  urine  in  the 
form  of  at  least  two  distinct  types  of  crystals,  i.  e.,  the  dumb-bell 


URINE.  345 


type  and  the  octahedral  type  (  Fig.  99,  below).  Either  form  may 
occur  in  the  sediment  of  neutral,  alkaline  or  acid  urine,  bill  both 
forms  are  found  most  frequently  in  urine  having  an  acid  reaction. 
Occasionally,  in  alkaline  urine,  the  octahedral  form  is  confounded 


Fig.  99. 


tP 


o  8 


03     * 


Calcium  Oxalate.     (Ogden.) 

with  "triple  phosphate"  crystals.  They  may  be  differentiated  from 
the  phosphate  crystals  by  the  fact  that  they  are  insoluble  in  acetic 
acid. 

The  presence  of  calcium  oxalate  in  the  urine  is  not  of  itself  a 
sign  of  any  abnormality,  since  it  is  a  constituent  of  normal  urine. 
It  is  increased  above  the  normal,  however,  in  such  pathological  con 
ditions  as  diabetes  mellitus,  in  organic  diseases  of  the  liver  and  in 
various  other  conditions  which  are  accompanied  by  a  derangement 
of  digestion  or  of  the  oxidation  mechanism,  such  as  occurs  in  cer- 
tain diseases  of  the  heart  and  lungs. 

Calcium  Carbonate. — Calcium  carbonate  crystals  form  a  typical 
constituent  of  the  urine  of  herbivorous  animals.  The}-  occur  less 
frequently  in  human  urine.  The  reaction  of  urine  containing  these 
crystals  is  nearly  always  alkaline,  although  they  may  occur  in  am- 
photeric or  in  slightly  acid  urine.  It  generally  crystallizes  in  the 
form  of  granules,  spherules  or  dumb-bells  (Fig.  100,  p.  346). 
The  crystals  of  calcium  carbonate  may  be  differentiated  from  cal- 
cium oxalate  by  the  fact  that  they  dissolve  in  acetic  acid  with  the 
evolution  of  carbon  dioxide  gas. 

Calcium  Phosphate  (Stellar  Phosphate). — Calcium  phosphate 
may  occur  in  the  urine  in  three  forms,  /'.  e.,  amorphous,  granular  or 
crystalline.  The  crystals  of  calcium  phosphate  arc  ordinarily 
pointed,  wedge-shaped  formations  which  may  occur  as  individual 
crystals  or  grouped  together  in  more  or  less  regularly  formed 
rosettes  (Fig.  76,  p.  224).     Acid  sodium  urate  crystals  1  Fig.  [02, 


346 


PHYSIOLOGICAL    CHEMISTRY. 


p.  348)  are  often  mistaken  for  crystals  of  calcium  phosphate.  We 
may  differentiate  between  these  two  crystalline  forms  by  the  fact 
that  acetic  acid  will  readily  dissolve  the  phosphate,  whereas  the  urate 
is  much  less  soluble  and  when  finally  brought  into  solution  and  re- 
crystallized  one  is  frequently  enabled  to  identify  uric  acid  crystals 

Fig.   100. 


Calcium  Carbonate. 


which  have  been  formed  from  the  acid  urate  solution.  The  clinical 
significance  of  the  occurrence  of  calcium  phosphate  crystals  in  the 
urinary  sediment  is  similar  to  that  of  "triple  phosphate"  (see 
page  344). 

Calcium  Sulphate. — Crystals  of  calcium  sulphate  are  of  quite 
rare  occurrence  in  the  sediment  of  urine.  Their  presence  seems  to 
be  limited  in  general  to  urines  which  are  of  a  decided  acid  reaction. 
Ordinarily  it  crystallizes  in  the  form  of  long,  thin,  colorless  needles 
or  prisms  (Fig.  95,  page  298)  which  may  be  mistaken  for  calcium 
phosphate  crystals.  There  need  be  no  confusion,  in  this  respect, 
however,  since  the  sulphate  crystals  are  insoluble  in  acetic  acid 
which  reagent  readily  dissolves  the  phosphate.  As  far  as  is  known 
their  occurrence  as  a  constituent  of  urinary  sediment  is  of  very 
little  clinical  significance. 

Uric  Acid. — Uric  acid  forms  a  very  common  constituent  of  the 
sediment  of  urines  which  are  acid  in  reaction.  It  occurs  in  more 
varied  forms  than  any  of  the  other  crystalline  sediments  (Plate  V, 
opposite  page  273,  and  Fig.  101,  page  347),  some  of  the  more  com- 
mon varieties  of  crystals  being  rhombic  prisms,  wedges,  dumb-bells, 


I'KI  \  I-.. 


whetstones,  prismatic  rosettes,  irregular  rectangular  or  hexagonal 
plates,  etc.  Crystals  of  pure  uric  acid  are  always  colorless  <  Fig. 
89,  page  274),  but  the  form  occurring  in  urinary  sediments  is  ira 
pure  and  under  the  microscope  appears  pigmented,  the  depth  of 

color  varying  from  light  yellow  to  a  dark  reddish-brown  according 
to  the  size  and  form  of  the  crystal. 

The  presence  of  a  considerable  uric  acid  sediment  does  not,  of 
necessity,  indicate  a  pathological  condition  or  a  urine  of  increased 
uric  acid  content,  since  this  substance  very  often  occurs  as  a  sedi 
ment  in  urines  whose  uric  acid  content  is  diminished  from  the  nor- 
mal merely  as  a  result  of  changes  in  reaction,  etc.  Pathologically, 
uric  acid  sediments  occur  in  gout,  acute  febrile  conditions,  chronic 
interstitial  nephritis,  etc.  If  the  microscopical  examination  is  not 
conclusive,  uric  acid  may  be  differentiated   from  other  crystalline 


Fig.  ioi. 


Various    Forms   of   Uric   Acid. 


1,  Rhombic  plates;  2,  whetstone  forms;  3,  3,  quadrate  forms;  4.  5,  prolonged  into 
points;  6,  8,  rosettes;  7,  pointed  bundles;  o,  barrel  forms  precipitated  by  adding 
hydrochloric  acid  to  urine. 

urinary  sediments  from  the  fact  that  it  is  soluble  in  alkalis,  alkali 
carbonates,  boiling  glycerol,  concentrated  sulphuric  acid  and  in  cer- 
tain organic  bases  such  as  ethylamine  and  piperidin.  It  also  re- 
sponds to  the  murexide  test  (see  page  274),  Selhff's  reaction  (see 
page  275)  and  to  Moreigne's  reaction  (see  p.  275). 

Urates. — The  urate  sediment  may  consist   of  a  mixture  of  the 


348 


PHYSIOLOGICAL    CHEMISTRY. 


urates  of  ammonium,  calcium,  magnesium,  potassium  and  sodium. 
The  ammonium  urate  may  occur  in  neutral,  alkaline  or  acid  urine, 
whereas  the  other  forms  of  urates  are  confined  to  the  sediments  of 
acid  urines.  Sodium  urate  occurs  in  sediments  more  abundantly 
than  the  other  urates.  The  urates  of  calcium,  magnesium  and  po- 
tassium are  amorphous  in  character,  whereas  the  urate  of  ammon- 
ium is  crystalline.  Sodium  urate  may  be  either  amorphous 
or  crystalline.  When  crystalline  it  forms  groups  of  fan-shaped 
clusters  or  colorless,  prismatic  needles  (Fig.  102,  below).  Am- 
monium urate  is  ordinarily  present  in  the  sediment  in  the  burr-like 
form  of  the  "thorn-apple"  crystal,  i.  e.,  yellow  or  reddish-brown 
spheres,  covered  with  sharp  spicules  or  prisms  (Plate  VI,  oppo- 
site).    The  urates  are  all  soluble  in  hydrochloric  acid  or  acetic 

Fig.   102. 


Acid  Sodium  Urate. 


acid  and  their  acid  solutions  yield  crystals  of  uric  acid  upon  stand- 
ing. They  also  respond  to  the  murexide  test.  The  clinical  sig- 
nificance of  urate  sediments  is  very  similar  to  that  of  uric  acid.  A 
considerable  sediment  of  amorphous  urates  does  not  necessarily 
indicate  a  high  uric  acid  content,  but  ordinarily  signifies  a  concen- 
trated urine  having  a  very  strong  acidity. 

Cystine. — Cystine  is  one  of  the  rarer  of  the  crystalline  urinary 
sediments.  It  has  been  claimed  that  it  occurs  more  often  in  the 
urine  of  men  than  of  women.  Cystine  crystallizes  in  the  form  of 
thin,  colorless,  hexagonal  plates  (Fig.  32,  p.  73,  and  Fig.  103, 
p.  349)  which  are  insoluble  in  water,  alcohol  and  acetic  acid  and 


PLATE  VI. 


Ammonium  Urate,  showing  Spherules  and  Thorn-apple-shaped  Crystals. 
(From  Ogden,  after  Peyer.) 


URINE.  349 

soluble  in  minerals  acids,  alkalis  and  especially  in  ammonia.  ( lystine 
may  be  identified  by  burning  it  upon  platinum  foil,  under  which 
condition  it  does  not  melt  but  yields  a  bluish-green  flame. 

Fig.  103. 


/ 


Cystine.     (Ogden.) 

Cholesterol. — Cholesterol  crystals  have  been  but  rarely  detected 
in  urinary  sediments.  When  present  they  probably  arise  from  a 
pathological  condition  of  some  portion  of  the  urinary  tract.  Crys- 
tals of  cholesterol  have  been  found  in  the  sediment  in  cystitis,  pye- 
litis, chyluria  and  nephritis.  Ordinarily  it  crystallizes  in  large  reg- 
ular and  irregular  colorless,  transparent  plates,  some  of  which  pos- 
sess notched  corners  (Fig.  42,  page  159).  Frequently,  instead  of 
occurring  in  the  sediment,  it  is  found  in  the  form  of  a  film  on  the 
surface  of  the  urine. 

Hippuric  Acid. — This  is  one  of  the  rarer  sediments  of  human 
urine.  It  deposits  under  conditions  similar  to  those  which  govern 
the  formation  of  uric  acid  sediments.  The  crystals,  which  are 
colorless  needles  or  prisms  (Fig.  92,  page  282)  when  pure,  are  in- 
variably pigmented  in  a  manner  similar  to  the  uric  acid  crystals 
when  observed  in  urinary  sediment  and  because  of  this  fact  are  fre- 
quently confounded  with  the  rarer  forms  of  uric  acid.  Hippuric 
acid  may  be  differentiated  from  uric  acid  from  the  fact  that  it  does 
not  respond  to  the  murexide  test  and  is  much  more  soluble  in  water 
and  in  ether.  The  detection  of  crystals  of  hippuric  acid  in  the  urine 
has  very  little  clinical  significance,  since  its  presence  in  the  sediment 
depends  in  most  instances  very  greatly  upon  the  nature  of  the  diet. 
It  is  particularly  prone  to  occur  in  the  sediment  after  the  ingestion 
of  certain  fruits  as  well  as  after  the  ingestion  of  benzoic  acid  1  see 
page  282). 

Leucine  and  Tyrosine. — Leucine  and  tyrosine  have  frequently 
been  detected  in  the  urine,  either  in  solution  or  as  a  sediment. 
Neither  of  them  occurs  in  the  urine  ordinarily  except  in  association 


350  PHYSIOLOGICAL    CHEMISTRY. 

with  the  other,  i.  e.,  whenever  leucine  is  detected  it  is  more  than 
probable  that  tyrosine  accompanies  it.  They  have  been  found  path- 
ologically in  the  urine  in  acute  yellow  atrophy  of  the  liver,  in  acute 
phosphorus  poisoning,  in  cirrhosis  of  the  liver,  in  severe  cases  of 
typhoid  fever  and  smallpox,  and  in  leukaemia.  In  urinary  sedi- 
ments leucine  ordinarily  crystallizes 
FlG'  I04-  ^mam.  m    characteristic    spherical    masses 

which  show  both  radial  and  concen- 
tric striations  and  are  highly  refrac- 
tive (Fig.  104,  p.  350).  Some  in- 
vestigators claim  that  these  crystals 
which  are  ordinarily  called  leucine 
are  in  reality,  generally  urates.  For 
the  crystalline  form  of  pure  leucine 
S>  obtained  as  a  decomposition  product 

Crystals  of  Impure  Leucine.  f  protejn  see  pig    2g    p>  y^       Tyro- 

sine  crystallizes  in  urinary  sediments 
in  the  well-known  sheaf  or  tuft  formation  (Fig.  23,  p.  72).  For 
other  tests  on  leucine  and  tyrosine  see  pages  83  and  84. 

Haematoidin  and  Bilirubin. — There  are  divergent  opinions  re- 
garding the  occurrence  of  these  bodies  in  urinary  sediment.  Each 
of  them  crystallizes  in  the  form  of  tufts  of  small  needles  or  in  the 
form  of  small  plates  which  are  ordinarily  yellowish-red  in  color 
(Fig.  41,  p.  153).  Because  of  the  fact  that  the  crystalline  form  of 
the  two  substances  is  identical  many  investigators  claim  them  to  be 
one  and  the  same  body.  Other  investigators  claim,  that  while  the 
crystalline  form  is  the  same  in  each  case,  that  there  are  certain 
chemical  differences  which  may  be  brought  out  very  strikingly  by 
properly  testing.  For  instance,  it  has  been  claimed  that  haematoidin 
may  be  differentiated  from  bilirubin  through  the  fact  that  it  gives 
a  momentary  color  reaction  (blue)  when  nitric  acid  is  brought  in 
contact  with  it,  and  further,  that  it  is  not  dissolved  on  treatment 
with  ether  or  potassium  hydroxide.  Pathologically,  typical  crystals 
of  haematoidin  or  bilirubin  have  been  found  in  the  urinary  sediment 
in  jaundice,  acute  yellow  atrophy  of  the  liver,  carcinoma  of  the  liver, 
cirrhosis  of  the  liver,  and  in  phosphorus  poisoning,  typhoid  fever 
and  scarlatina. 

Magnesium  Phosphate. — Magnesium  phosphate  crystals  occur 
rather  infrequently  in  the  sediment  of  urine  which  is  neutral,  alka- 
line or  feebly  acid  in  reaction.  It  ordinarily  crystallizes  in  elon- 
gated, highly  refractive,  rhombic  plates  which  are  soluble  in  acetic 
acid. 


URINE.  3  5  I 

Indigo. — Indigo  crystals  are  frequently  found  in  urine  which  has 
undergone  alkaline  fermentation.  The)  result  from  the  breaking 
down  of  indoxyl-sulphates  or  indoxyl-glycuronates.  Ordinarily 
indigo  deposits  as  dark  blue  stellate  needles  or  occurs  as  amorphous 
particles  or  broken  fragments.  These  crystalline  or  amorphous 
forms  may  occur  in  the  sedimenl  or  may  form  a  blue  film  on  the  sur- 
face of  the  urine.  Indigo  crystals  generally  occur  in  urine  which 
is  alkaline  in  reaction,  but  they  have  been  detected  in  acid  urine. 

Xanthine. — Xanthine  is  a  constituent  of  normal  urine  hut  is 
found  in  the  sediment  in  crystalline  form  very  infrequently,  and  then 
only  in  pathological  urine.  When  present  in  the  sediment  xanthine 
generally  occurs  in  the  form  of  whetstone-shaped  crystals  some- 
what similar  in  form  to  the  whetstone  variety  of  uric  acid  crystal. 
They  may  be  differentiated  from  uric  acid  by  the  great  ease  with 
which  they  may  be  brought  into  solution  in  dilute  ammonia  and  on 
applying  heat.  Xanthine  may  also  form  urinary  calculi.  The 
clinical  significance  of  xanthine  in  urinary  sediment  is  not  well 
understood. 

Melanin. — Melanin  is  an  extremely  rare  constituent  of  urinary 
sediments.  Ordinarily  in  melanuria  the  melanin  remains  in  solu- 
tion;  if  it  separates  it  is  generally  held  in  suspension  as  fine  amor- 
phous granules. 

(b)  Organized  Sediments. 

Epithelial  cells. 
Pus  cells. 

'  Hyaline. 

Granular. 

Epithelial. 
Casts.  -|  Blood. 

Fatty. 

Waxy. 
_  Pus. 
Cylindroids. 
Erythrocytes. 
Spermatozoa. 
Urethral  filaments. 
Tissue  debris. 
Animal  parasites. 
Micro-organisms. 
Fibrin. 
Foreign  substances  due  to  contamination. 


352 


PHYSIOLOGICAL    CHEMISTRY. 


Epithelial  Cells.---The  detection  of  a  certain  number  of  these 
cells  in  urinary  sediment  is  not,  of  itself,  a  pathological  sign,  since 
they  occur  in  normal  urine.  However,  in  certain  pathological  con- 
ditions they  are  greatly  increased  in  number,  and  since  different 
areas  of  the  urinary  tract  are  lined  with  different  forms  of  epithelial 
cells,  it  becomes  necessary,  when  examining  urinary  sediments,  to 
note  not  only  the  relative  number  of  such  cells,  but  at  the  same  time 
to  carefully  observe  the  shape  of  the  various  individuals  in  order 
to  determine,  as  far  as  possible,  from  what  portion  of  the  tract  they 
have  been  derived.  Since  the  different  layers  of  the  epithelial  lin- 
ing are  composed  of  cells  different  in  form  from  those  of  the  as- 
sociated layers,  it  is  evident  that  a  careful  microscopical  examina- 
tion of  these  cells  may  tell  us  the  particular  layer  which  is  being 
desquamated.  It  is  frequently  a  most  difficult  undertaking,  how- 
ever, to  make  a  clear  differentiation  between  the  various  forms  of 
epithelial  cells  present  in  a  sediment.  If  skilfully  done,  such  a 
microscopical  differentiation  may  prove  to  be  of  very  great  diag- 
nostic aid. 

The  principal  forms  of  epithelial  cells  met  with  in  urinary  sedi- 
ments are  shown  in  Fig.   105,  below. 

Fig.  105. 


Epithelium  from  Different  Areas  of  the  Urinary  Tract. 

a,  Leucocyte  (for  comparison)  ;  b,  renal  cells  ;  c,  superficial  pelvic  cells ;  d,  deep 
pelvic  cells  ;  e,  cells  from  calices  ;  /,  cells  from  ureter  ;  g,  g,  g,  g,  gt  squamous  epi- 
thelium from  the  bladder ;  h,  h,  neck-of-bladder  cells ;  i,  epithelium  from  prostatic 
urethra ;  k,  urethral  cells  ;  I,  I,  scaly  epithelium  ;  m,  m' ,  cells  from  seminal  passages  ; 
n,  compound  granule  cells ;  o,  fatty  renal  cell.     (Ogden.) 


URINE. 


353 


Pus  Cells. —  Pus  corpuscles  or  leucocytes  are  presenl  in  extremely 
small  numbers  in  normal  urine.  Any  considerable  increase  in  the 
number,  however,  ordinarily  denotes  a  pathological  condition,  -en 
erally  an  acute  or  chronic  inflammatory  condition  of  some  portion 
of  the  urinary  tract.  The  sudden  appearance  of  a  large  amount  of 
pus  in  a  sediment  denotes  the  opening  of  an  abscess  into  the  urinary 
tract.  Other  form  elements,  such  as  epithelial  cells,  cast.,  etc., 
ordinarily  accompany  pus  corpuscles  in  urinary  sediment  and  a 
careful  examination  of  these  associated  elements  is  necessary  in 
order  to  form  a  correct  diagnosis  as  to  the  origin  of  the  pus.  Pro- 
tein is  always  present  in  urine  which  contains  pus. 


Fig.   106. 


Pus  Corpusci.es.     (.After  Ultsmann.) 

i,  Normal;  2,  showing  amoeboid  movements;  3,  nuclei  rendered  distinct  by  acetic  acid 
4,  as  observed  in  chronic  pyelitis  ;  5,  swollen  by  ammonium  carbonate. 


The  appearance  which  pus  corpuscles  exhibit  under  the  micro- 
scope depends  greatly  upon  the  reaction  of  the  urine  containing 
them.  In  acid  urine  they  generally  present  the  appearance  of 
round,  colorless  cells  composed  of  refractive,  granular  protoplasm, 
and  may  frequently  exhibit  amoeboid  movements,  especially  if  the 
slide  containing  them  be  warmed  slightly.  They  are  nucleated 
(one  or  more  nuclei),  the  nuclei  being  clearly  visible  only  upon 
treating  the  cells  with  water,  acetic  acid  or  some  other  suitable 
reagent.  In  urine  which  has  a  decided  alkaline  reaction,  on  the 
other  hand,  the  pus  corpuscles  are  often  greatly  degenerated.  They 
may  be  seen  as  swollen,  transparent  cells,  which  exhibit  no  granular 
24 


354 


PHYSIOLOGICAL    CHEMISTRY. 


structure  and  as  the  process  of  degeneration  continues  the  cell  out- 
line ceases  to  be  visible,  the  nuclei  fade,  and  finally  only  a  mass  of 
debris  containing  isolated  nuclei  and  an  occasional  cell  remains. 

It  is  frequently  rather  difficult  to  make  a  differentiation  between 
pus  corpuscles  and  certain  types  of  epithelial  cells  which  are  similar 
in  form.  Such  confusion  may  be  avoided  by  the  addition  of  iodine 
solution  (I  in  KI),  a  reagent  which  stains  the  pus  corpuscles  a  deep 
mahogany-brown  and  transmits  to  the  epithelial  cells  a  light  yellow 
tint.  The  test  proposed  by  Vitali  often  gives  very  satisfactory  re- 
sults. This  simply  consists  in  acidifying  the  urine  (if  alkaline) 
with  acetic  acid,  then  filtering,  and  treating  the  sediment  on  the 
filter  paper  with  freshly  prepared  tincture  of  guaiac.  The  presence 
of  pus  in  the  sediment  is  indicated  if  a  blue  color  is  observed. 
Large  numbers  of  pus  corpuscles  are  present  in  the  urinary  sedi- 
ment in  gonorrhoea,  leucorrhcea,  chronic  pyelitis  and  in  abscess  of 
the  kidney. 

Fig.  107. 


Hyaline   Casts. 
One  cast  is  impregnated  with  four  renal   cells. 


Casts. — These  are  cylindrical  formations,  which  originate  in  the 
uriniferous  tubules  and  are  forced  out  by  the  pressure  of  the  urine. 
They  vary  greatly  in  size  but  in  nearly  every  instance  they  possess 
parallel  sides  and  rounded  ends.     The  finding  of  casts  in  the  urine 


URINE. 


355 


is  very  important  because  of  the  fact  thai  they  generally  indicate 
some  kidney  disorder;  if  albumin  accompanies  the  casts  the  indica- 
tion is  much  accentuated.     Casts  have  been  classified  according  to 

their  microscopical  characteristics  as  follows:  (a)  Hyaline.  </<j 
granular,  (c)  epithelial,  (d)  blood,  (e)  fatty,  (/)  waxy,  (g)  pus. 
(a)  Hyaline  Casts. — These  are  composed  of  a  basic  material 
which  is  transparent,  homogeneous  and  very  light  in  color  i  Fig. 
107,  p.  354).     In  fact,  chiefly  because  of  these  physical  properties, 


Fig.   108. 


Granular  Casts.     (After  Peyer.) 


they  are  the  most  difficult  form  of  renal  casts  to  detect  under  the 
microscope.  Frequently  such  casts  are  impregnated  with  deposits 
of  various  forms,  such  as  erythrocytes,  epithelial  cells,  fat  globules, 
etc.,  thus  rendering  the  form  of  the  cast  more  plainly  visible. 
Staining  is  often- resorted  to  in  order  to  render  the  shape  and  char- 
acter of  the  cast  more  easily  determined.  Ordinary  iodine  solution 
(I  in  KI)  may  be  used  in  this  connection ;  many  of  the  aniline  dyes 
are  also  in  common  use  for  this  purpose,  e.  g.3  gentian-violet,  Bis- 
marck-brown, methylene-blue.  fuchsin  and  eosin.  Generally,  but 
not  always,  albumin  is  present  in  urine  containing  hyaline  casts. 
Hyaline  casts  are  common  to  all  kidney  disorders,  but  occur  par- 
ticularly in  the  earliest  and  recovering  stages  of  parenchymatous 
nephritis  and  in  interstitial  nephritis. 


356 


PHYSIOLOGICAL    CHEMISTRY. 


(b)  Granular  Casts.— The  common  hyaline  material  is  ordinarily 
the  basic  substance  of  this  form  of  cast 


The  granular  material 


Fig.  109. 


Granular  Casts. 

a,  Finely  granular ;  b,  coarsely 

granular. 


Fig.  no. 


a?VS%-, 


generally  consists  of  albumin,  epithelial  cells,  fat  or  disintegrated 
erythrocytes  or  leucocytes,  the  character  of  the  cast  varying  accord- 
ing to  the  nature  and  size  of  the  granules  (Fig.  108,  page  355,  and 
Fig.  109,  above).     Thus  we  have  casts  of  this  general  type  classi- 


FlG. 


5lood,  Pus,  Hyaline  and  Epithelial  Casts. 

a,  Blood  casts  ;   b,  pus  cast ;  c,  hyaline  cast  impregnated    with  renal  cells  ; 

d,  epithelial  casts. 


URINE. 


357 


Fatty  Casts.     (After  Peyer.) 


Fig.   113. 


Fatty  and  Waxy   Casts. 
a,  Fatty  casts  ;   b,  waxy  casts. 


35^  PHYSIOLOGICAL    CHEMISTRY. 

fied  as  finely  granular  and  coarsely  granular  casts.  Granular  casts, 
and  in  particular  the  finely  granular  types,  occur  in  the  sediment  in 
practically  every  kidney  disorder  but  are  probably  especially  char- 
acteristic of  the  sediment  in  inflammatory  disorders. 

(c)  Epithelial  Casts. — These  are  casts  bearing  upon  their  sur- 
face epithelial  cells  from  the  lining  of  the  uriniferous  tubules  (Fig. 
no,  p.  356).  The  basic  material  of  this  form  of  cast  may  be  hya- 
line or  granular  in  nature.  Epithelial  casts  are  particularly  abun- 
dant in  the  urinary  sediment  in  acute  nephritis. 

(d)  Blood  Casts.- — Casts  of  this  type  may  consist  of  erythrocytes 
borne  upon  a  hyaline  or  a  fibrinous  basis  (Fig.  in,  p.  356).  The 
occurrence  of  such  casts  in  the  urinary  sediment  denotes  renal 
hemorrhage  and  they  are  considered  to  be  especially  characteristic 
of  acute  diffuse  nephritis  and  acute  congestion  of  the  kidney. 

(<?)  Fatty  Casts. — Fatty  casts  may  be  formed  by  the  deposition 
of  fat  globules  or  crystals  of  fatty  acid  upon  the  surface  of  a  hya- 
line or  granular  cast  (Fig.  112,  p.  357).  In  order  to  constitute  a 
true  fatty  cast  the  deposited  material  must  cover  the  greater  part  of 
the  surface  area  of  the  cast.  The  presence  of  fatty  casts  in  urinary 
sediment  indicates  fatty  degeneration  of  the  kidney;  such  casts  are 
particularly  characteristic  of  subacute  and  chronic  inflammations 
of  the  kidney. 

(/)    Waxy  Casts. — -These  casts  possess  a  basic  substance  similar 

Fig.  114. 


Cylindroids.     (After  Peyer.) 


URINK. 


359 


to  that  which  enters  into  the  foundation  of  the  hyaline  form  of 
cast.  In  common  with  the  hyaline  type  they  arc  colorless,  refractive 
bodies  but  differ  From  this  form  of   cast  in  being,  in  general,  of 

greater  length  and  diameter  and  possessing  sharper  outlines  and  a 
light  yellow  color  (Fig.  113,  p.  357).  Such  casts  occur  in  several 
forms  of  nephritis  but  do  not  appeal-  to  characterize  any  particular 
type  of  the  disorder  except  amyloid  disease,  in  which  they  are  rather 
common. 

(g)  Pus  Costs. — Casts  whose  surface  is  covered  with  pus  cells 
or  leucocytes  are  termed  pus  casts  (Fig.  ill,  p.  356).  They  are  fre- 
quently mistaken  for  epithelial  casts.  The  differentiation  between 
these  two  types  is  made  very  simple  however  by  treating  the  cast 
with  acetic  acid  which  causes  the  nuclei  of  the  leucocytes  to  become 
plainly  visible.  The  true  pus  cast  is  quite  rare  and  indicates  renal 
suppuration. 

Cylindroids. — These  formations  may  occur  in  normal  or  patho- 
logical urine  and  have  no  particular  clinical  significance.  They 
are  frequently  mistaken  for  true  casts,  especially  the  hyaline  type, 
but  they  are  ordinarily  flat  in  structure  with  a  rather  smaller  diam- 

Fig.    115. 


CRENATED    ErI   fHROI    1   M   -. 


eter  than  casts,  may  possess  forked  or  branching  ends  and  are  not 
composed  of  homogeneous  material  as  are  the  hyaline  casts.  Such 
''false  casts"  may  become  coated  with  urates,  in  which  event  they 
appear  granular  in  structure.  The  basic  substance  of  cylindroids  is 
often  the  nucleop'-otein  of  the  urine  (  see  Fig.  1 14.  page  358). 


360 


PHYSIOLOGICAL    CHEMISTRY. 


Erythrocytes. — These  form  elements  are  present  in  the  urinary 
sediment  in  various  diseases.  They  may  appear  as  the  normal  bi- 
concave, yellow  erythrocyte  (Plate  IV,  opposite  page  184)  or  may 
exhibit  certain  modifications  in  form  such  as  the  crenated  type 
(Fig.  115,  p.  359)  which  is  often  seen  in  concentrated  urine.  Un- 
der different  conditions  they  may  become  swollen  sufficiently  to 
entirely  erase  the  biconcave  appearance  and  may  even  occur  in  the 
form  of  colorless  spheres  having  a  smaller  diameter  than  the  original 
disc-shaped  corpuscles.  Erythrocytes  are  found  in  urinary  sedi- 
ment in  hemorrhage  of  the  kidney  or  of  the  urinary  tract,  in 
traumatic  hemorrhage,  hemorrhage  from  congestion  and  in  hemor- 
rhagic diathesis. 

Spermatozoa. — Spermatozoa  may  be  detected  in  the  urinary 
sediment  in  diseases  of  the  genital  organs,  as  well  as  after  coitus, 
nocturnal  emissions,  epileptic  and  other  convulsive  attacks  and  some- 

Fig.  116. 


Human  Spermatozoa. 


times  in  severe  febrile  disorders,  especially  in  typhoid  fever.  In 
form  they  consist  of  an  oval  body,  to  which  is  attached  a  long, 
delicate  tail  (Fig.  116,  above).  Upon  examination  they  may  show 
motility  or  may  be  motionless. 

Urethral  Filaments. — These  are  peculiar  thread-like  bodies 
which  are  sometimes  found  in  urinary  sediment.  They  may  oc- 
casionally be  detected  in  normal  urine  and  pathologically  are  found 
in  the  sediment  in  acute  and  chronic  gonorrhoea  and  in  urethror- 
rhcea.     The  ground-substance  of  these  urethral  filaments  is  in  part, 


URINK.  361 

at  least,  similar  to  that  of  the  cylindroids  (see  page  359).  The 
urine  first  voided  in  the  morning  is  best  adapted  fi  >r  the  examinatii  »n 
for  filaments.     These  filaments  may  ordinarily  be  removed  by  a 

pipette  since  they  arc  generally  macroscopic. 

Tissue  Debris. — Masses  of  cells  or  fragments  of  tissue  are  fre- 
quently found  in  the  urinary  sediment.  They  may  be  found  in  the 
sediment  in  tubercular  affections  of  the  kidney  and  urinary  tract 
or  in  tumors  of  these  organs.  Ordinarily  it  is  necessary  to  make  a 
histological  examination  of  such  tissue  fragments  before  coming 
to  a  final  decision  as  to  their  origin. 

Animal  Parasites. — -The  cysts,  hooklets  and  membrane  shreds 
of  ecJiinococci  are  sometimes  found  in  the  urinary  sediments. 
Other  animal  organisms  which  are  more  rarely  met  with  in  the 
urine  are  embryos  of  the  Filaria  sanguinis  and  eggs  of  the  Distoma 
haematobium  and  Ascarides.  Animal  parasites  in  general  occur 
most  frequently  in  the  urine  in  tropical  countries. 

Micro-Organisms. — Bacteria  as  well  as  yeasts  and  moulds  are 
frequently  detected  in  the  urine.  Both  the  pathogenic  and  non- 
pathogenic forms  of  bacteria  may  occur.  The  non-pathogenic 
forms  most  frequently  observed  are  micrococcus  urea,  bacillus 
ureas,  and  staphylococcus  urea  liquefaciens.  Of  the  pathogenic 
forms  many  have  been  obsrved,  e.  g.,  Bacterium  Coli,  typhoid  ba- 
cillus, tubercle  bacillus,  gonococcus,  bacillus  pyocyaneus  and  pro  tens 
vulgaris.  Yeast  and  moulds  are  most  frequently  met  with  in  dia- 
betic urine. 

Fibrin. — Following  hematuria,  fibrin  clots  are  occasionally  ob- 
served in  the  urinary  sediment.  They  are  generally  of  a  semi- 
gelatinous  consistency  and  of  a  very  light  color,  and  when  examined 
under  the  microscope  they  are  seen  to  be  composed  of  bundles  of 
highly  refractive  fibers  which  run  parallel. 

Foreign  Substances  Due  to  Contamination. — Such  foreign 
substances  as  fibers  of  silk,  linen  or  wool;  starch  granules,  hair,  fat 
and  sputum,  as  well  as  muscle  fibers,  vegetable  cells  and  food  par- 
ticles are  often  found  in  the  urine.  Care  should  be  taken  that 
these  foreign  substances  are  not  mistaken  for  any  of  the  true  sedi- 
mentary constituents  already  mentioned. 


CHAPTER   XXI. 

URINE:    CALCULI. 

Urinary  calculi,  also  called  concretions,  or  concrements  are  solid 
masses  of  urinary  sediment  formed  in  some  part  of  the  urinary 
tract.  They  vary  in  shape  and  size  according-  to  their  location,  the 
smaller  calculi  termed  sand  or  gravel  in  general  arising  from  the 
kidney  or  the  pelvic  portion  of  the  kidney,  whereas  the  large  calculi 
are  ordinarily  formed  in  the  bladder.  There  are  two  general 
classes  of  calculi  as  regards  composition,  i.  e.,  simple  and  compound. 
The  simple  form  is  made  up  of  but  a  single  constituent  whereas  the 
compound  type  contains  two  or  rfiore  individual  constituents.  The 
structural  plan  of  most  calculi  consists  of  an  arrangement  of  con- 
centric rings  about  a  central  nucleus,  the  number  of  rings  frequently 
being  dependent  upon  the  number  of  individual  constituents  which 
enter  into  the  structure  of  the  calculus.  In  case  two  or  more  cal- 
culi unite  to  form  a  single  calculus  the  resultant  body  will  obviously 
contain  as  many  nuclei  as  there  were  individual  calculi  concerned 
in  its  construction.  Under  certain  conditions  the  growth  of  a  cal- 
culus will  be  principally  in  only  one  direction,  thus  preventing  the 
nucleus  from  maintaining  a  central  location.  The  qualitative  com- 
position of  urinary  calculi  is  dependent,  in  great  part,  upon  the  re- 
action of  the  urine  e.  g.,  if  the  reaction  of  the  urine  is  acid  the 
calculi  present  will  be  composed,  in  great  part  at  least,  of  substances 
that  are  capable  of  depositing  in  acid  urine. 

According  to  Ultzmann,  out  of  545  cases  of  urinary  calculus, 
uric  acid  and  urates  formed  the  nucleus  in  about  81  per  cent  of  the 
cases;  earthy  phosphates  in  about  9  per  cent;  calcium  oxalate  in 
about  6  per  cent ;  cystine  in  something  over  1  per  cent,  while  in  about 
3  per  cent  of  the  cases  some  foreign  body  comprised  the  nucleus. 

In  the  chemical  examination  of  urinary  calculi  the  most  valuable 
data  are  obtained  by  subjecting  each  of  the  concentric  layers  of  the 
calculus  to  a  separate  analysis.  Material  for  examination  may  be 
conveniently  obtained  by  sawing  the  calculus  carefully  through  the 
nucleus,  then  separating  the  various  layers  or  by  scraping  off  from 
each  layer  (without  separating  the  layers)  enough  powder  to  con- 
duct the  examination  as  outlined  in  the  scheme  (see  page  364). 

362 


URINE.  363 

Varieties  of  Calculus. 

Uric  Acid  and  Urate  Calculi. — Uric  acid  and  urates  constitute 
the  nuclei  of  a  large  proportion  ("8r  per  cent)  of  urinary  concre 
tions.     Such  stones  are  always  colored,  the  tint  varying  from  a  pale 
yellow  to  a  brownish-red.     The  surface  of  such  calculi  is  generally 
smooth  but  it  may  be  rough  and  uneven. 

Phosphatic  Calculi. — Ordinarily  these  concretions  consist  prin- 
cipally of  ''triple  phosphate"  and  other  phosphates  of  the  alkaline 
earths,  with  very  frequent  admixtures  of  urates  and  oxalates. 
The  surface  of  such  calculi  is  generally  rough  but  may  occasionally 
be  rather  smooth.  The  calculi  are  somewhat  variable  in  color,  ex- 
hibiting gray,  white  or  yellow  tints  under  different  conditions. 
When  composed  of  earthy  phosphates  the  calculi  are  character- 
ized by  their  friability. 

Calcium  Oxalate  Calculi. — This  is  the  hardest  form  of  calculus 
to  deal  with,  and  is  rather  difficult  to  crush.  They  ordinarily  occur 
in  two  general  forms,  i.  c,  the  small,  smooth  concretion  which  is 
characterized  as  the  hemp-seed  calculus  and  the  medium-sized  or 
large  stone  possessing  an  extremely  uneven  surface  which  is  gen- 
erally classed  as  a  mulberry  calculus.  This  roughened  surface  of 
the  latter  form  of  calculus  is  due,  in  many  instances,  to  protruding 
calcium  oxalate  crystals  of  the  octahedral  type. 

Calcium  Carbonate  Calculi. — Calcium  carbonate  concretions 
are  quite  common  in  herbivorous  animals  but  of  exceedingly  rare 
occurrence  in  man.  They  are  generally  small,  white  or  grayish 
calculi,  spherical  in  form  and  possess  a  hard,  smooth  surface. 

Cystine  Calculi. — The  cystine  calculus  is  a  rare  variety  of  cal- 
culus. Ordinarily  they  occur  as  small,  smooth,  oval  or  cylindrical 
concretions  which  are  white  or  yellow  in  color  and  of  a  rather  soft 
consistency. 

Xanthine  Calculi.- — -This  form  of  calculus  is  somewhat  more 
rare  than  the  cystine  type.  The  color  may  vary  from  white  to 
brownish-yellow.  Very  often  uric  acid  and  urates  are  associated 
with  xanthine  in  this  type  of  calculus.  Upon  rubbing  a  xanthine 
calculus  it  has  the  property  of  assuming  a  wax-like  appearance. 

Urostealith  Calculi. — This  form  of  calculus  is  extremely  rare. 
Such  concretions  are  composed  principally  of  fat  and  fatty  acid. 
When  moist  they  are  soft  and  elastic  but  when  dried  they  become 
brittle.     Urostealiths  are  generally  light  in  color. 

Fibrin  Calculi. — Fibrin  calculi  are  produced  in  the   proce- 


364 


PHYSIOLOGICAL    CHEMISTRY. 


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URINE.  365 

blood  coagulation  within  the  urinary  tract.  They  frequently  occur 
as  nuclei  of  other  forms  of  calculus.     They  arc  rarely  found. 

Cholesterol  Calculi. — An  extremely  rare  form  of  calculus  some- 
what resembling'  the  cystine  type. 

Indigo  Calculi. — Indigo  calculi  are  extremely  rare,  only  two 
cases  having  been  reported.  One  of  these  indigo  calculi  is  on  ex- 
hibition in  the  museum  of  Jefferson  Medical  College  of  Philadel- 
phia. 

The  scheme,  proposed  by  Heller  and  given  on  page  364,  will  be 
found  of  much  assistance  in  the  chemical  examination  of  urinary 
calculi. 


CHAPTER    XXII. 

URINE:   QUANTITATIVE  ANALYSIS. 
I.     Protein. 

i.  Scherer's  Coagulation  Method. — The  content  of  coagulable 
protein  may  be  accurately  determined  as  follows :  Place  50  c.c.  of 
urine  in  a  small  beaker  and  raise  the  temperature  of  the  fluid  to 
about  400  C.  upon  a  water-bath.  Add  dilute  acetic  acid,  drop  by 
drop,  to  the  warm  urine,  to  precipitate  the  protein  which  will  sep- 
arate in  a  flocculent  form.  Care  should  be  taken  not  to  add  too 
much  acid;  ordinarily  less  than  twenty  drops  is  sufficient.  The 
temperature  of  the  water  in  the  water-bath  should  now  be  raised  to 
the  boiling-point  and  maintained  there  for  a  few  minutes  in  order 
to  insure  the  complete  coagulation  of  the  protein  present.  Now 
filter  the  urine1  through  a  previously  washed,  dried  and  weighed 
filter  paper,  wash  the  precipitated  protein,  in  turn,  with  hot  water, 
95  per  cent  alcohol  and  with  ether,  and  dry  the  paper  and  precipitate, 
to  constant  weight,  in  an  air-bath  at  no°  C.  Subtract  the  weight 
of  the  filter  paper  from  the  combined  weight  of  the  paper  and 
precipitate  and  calculate  the  percentage  of  protein  in  the  urine 
specimen. 

Calculation. — To  determine  the  percentage  of  protein  present  in 
the  urine  under  examination,  multiply  the  weight  of  the  precipitate, 
expressed  in  grams,  by  2. 

2.  Esbach's  Method.- — This  method  depends  upon  the  precipi- 
tation of  protein  by  Esbach's  reagent2  and  the  apparatus  used  in 
the  estimation  is  Esbach's  albuminometer  (Fig.  117,  p.  367).  In 
making  a  determination  fill  the  albuminometer  to  the  point  U  with 
urine,  then  introduce  the  reagent  until  the  point  R  is  reached.  Now 
stopper  the  tube,  invert  it  slowly  several  times  in  order  to  insure  the 

1  If  it  is  desired  the  precipitate  may  be  filtered  off  on  an  unweighed  paper,  and 
its  nitrogen  content  determined  by  the  Kjeldahl  method  (see  p.  381).  In  order 
to  arrive  at  correct  figures  for  the  protein  content  it  is  then  simply  necessary  to 
multiply  the  total  nitrogen  content  by  6.25  (see  p.  412).  Correction  should  be 
made  for  the  nitrogen  content  of  the  filter-paper  used  unless  this  factor  is 
negligible. 

2  Esbach's  reagent  is  prepared  by  dissolving  10  grams  of  picric  acid  and  20 
grams  of  citric  acid  in   1  liter  of  water. 

366 


IKIXi::    (jl'A.VTITATIVK    ANALYSIS. 


thorough  mixing-  of  the  fluids  and  stand  the  tube  aside  for  24  hours. 

Creatinine,  resin  acids,  etc.,  are  precipitated  in  this  method,  and  for 
this  and  other  reasons  it  is  not  as  accurate  as  the  coagulation  method. 
It  is.  however,  extensively  used  clinical]}. 

Calculation. — The   graduations   on    the   albuminpmeter    indicate 

grams  of  protein  per  liter  of  urine.  Thus,  if  the  protein  precipi- 
tate is  level  with  the  figure  7.  of  the  graduated 
scale  this  denotes  that  the  urine  examined  con- 
tains 3  grams  of  protein  to  the  liter.  To  express 
the  amount  of  protein  in  per  cent  simply  move 
the  decimal  point  one  place  to  the  left.  In  the 
case  under  consideration  the  urine  contains  0.3 
per  cent  of  protein. 


II.     Dextrose. 

1.   Fehling's   Method. — Place    to  c.c.   of   the 


—tt 


urine  under  examination  in  a  100  c.c.  volumetric 
flask  and  make  the  volume  up  to  100  c.c.  with 
distilled  water.  Thoroughly  mix  this  diluted 
urine,  by  pouring  it  into  a  beaker  and  stirring 
with  a  glass  rod,  then  transfer  a  portion  of  it 
to  a  burette  which  is  properly  supported  in  a 
clamp. 

Now  place  10  c.c.  of  Fehling's  solution1  in  a 
small  beaker,  dilute  it  with  approximately  40  c.c. 
of  distilled  water,  heat  to  boiling,  and  observe 
whether  decomposition  of  the  Fehling's  solution 
itself  has  occurred  as  indicated  by  the  production 
of  a  turbidity.  If  such  turbidity  is  produced  the 
Fehling's  solution  is  unfit  for  use.  Clamp  the 
burette  containing  the  diluted  urine  immediately 
over  the  beaker  and  carefully  allow  from  0.5  to  1 
c.c.  of  the  diluted  urine  to  flow  into  the  boiling 
Fehling's  solution.  Bring  the  solution  to  the  boil- 
ing-point after  each  addition  of  urine  and  continue 
running  in  the  urine  from  the  burette.  0.5-1  c.c.  at  a  time,  as  indi- 
cated, until  the  Fehling's  solution  is  completely  reduced,  i.  e..  until 
all  the  cupric  oxide  in  solution  has  been  precipitated  as  cuprous  oxide. 
This  point  will  be  indicated  by  the  absolute  disappearance  of  all  blue 
color.  When  this  end-point  is  reached  note  the  number  of  cubic  centi- 

1  Directions  for  the  preparation  of  Fehling's  solution  are  given  in  a  note  at 

the  bottom  of  page  308. 


■R 


Esbach's  Albumi- 
nometer.   i  i 


368  PHYSIOLOGICAL    CHEMISTRY. 

meters  of  diluted  urine  used  in  the  process  and  calculate  the  per- 
centage of  dextrose  present,  in  the  sample  of  urine  analyzed,  ac- 
cording to  the  method  given  below. 

This  is  a  very  satisfactory  method,  the  main  objection  to  its  use 
being  the  uncertainty  attending  the  determination  of  the  end-reac- 
tion, i.  e.,  the  difficulty  with  which  the  exact  point  where  the  blue 
color  -finally  disappears  is  noted.  Several  means  of  accurately  fix- 
ing this  point  have  been  suggested  but  they  are  practically  all  open 
to  objection.  As  good  a  "  check  "  as  any,  perhaps,  is  to  filter  a  few 
drops  of  the  solution,  through  a  double  paper,  after  the  blue  color 
has  apparently  disappeared,  acidify  the  filtrate  with  acetic  acid  and 
add  potassium  ferrocyanide.  If  the  copper  of  the  Fehling's  solu- 
tion has  been  completely  reduced,  there  will  be  no  color  reaction, 
whereas  the  production  of  a  brown  color  indicates  the  presence  of 
unreduced  copper.  Harrison  has  recently  suggested  the  follow- 
ing procedure  to  determine  the  exact  end-point:  To  about  I  c.c. 
of  a  starch  iodide  solution1  in  a  test-tube  add  2-3  drops  of  acetic 
acid  and  introduce  into  the  acidified  mixture  1-2  drops  of  the 
solution  to  be  tested.  Unreduced  copper  will  be  indicated  by  the 
production  of  a  purplish-red  or  blue  color  due  to  the  liberation  of 
iodine. 

It  is  ordinarily  customary  to  make  at  least  three  determinations 
by  Fehling's  method  before  coming'  to  a  final  conclusion  regarding 
the  sugar  content  of  the  urine  under  examination. 

Calculation. — Ten  c.c.  of  Fehling's  solution  is  completely  reduced 
by  0.05  gram  of  dextrose.2  If  y  represents  the  number  of  cubic 
centimeters  of  undiluted  urine  (obtained  by  dividing  the  burette 
reading  by  10)  necessary  to  reduce  the  10  c.c.  of  Fehling's  solu- 
tion, we  have  the  following  proportion : 

3; :  0.05  : :  too  :  x  (percentage  of  dextrose) . 
2.  Benedict's  Method. — To  30  c.c.  of  Benedict's  solution3  in 

1  The  starch-iodide  solution  may  be  prepared  as  follows:  Mix  0.1  gram  of 
starch  with  cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into 
75-100  c.c.  of  boiling  water,  stirring  continuously.  Cool  the  starch  paste,  add 
20-25  grams  of  potassium  iodide  and  dilute  the  mixture  to  250  c.c.  This  solution 
deteriorates  upon  standing,  and  therefore  must  be  freshly  prepared  as  needed. 

2  The  values  for  certain  other  sugars  are  as  follows : 

Lactose    0.0676  gram. 

Maltose    0.074    gram. 

Invert  sugar 0.0475  gram. 

3  Benedict's  solution  used  in  the  quantitative  determination  of  sugar  consists  of 
three  separate  solutions.     The  cupric  sulphate  solution  and  the  alkaline  tartrate 


urine:  QUANTITATIVE   ANALYSIS.  $69 

a  small  beaker  add  from  2.^  grams  to  5  grams  of  anhydrous  -odium 
carbonate1  and  beat  tbe  mixture  to  boiling  over  a  wire  gauze  until 
tbe  carbonate  lias  been  brought  into  solution. 

Place  the  urine  under  examination  in  a  burette  and  run  it  into 
the  hot  Benedict  solution  rather  rapidly2  until  the  formation  of  a 
heavy  chalk-white  precipitate  is  noted  and  the  blue  color  of  the  solu- 
tion lessens  perceptibly  in  its  intensity.  From  this  point  in  the  de- 
termination from  2  to  10  drops3  of  the  urine  should  be  run  into 
the  boiling  Benedict  solution  at  one  time,  boiling  the  solution  vig- 
orously for  about  15  seconds  after  each  addition.  Complete  re- 
duction of  the  copper  is  indicated  here  as  in  Fehling's  original 
method,  by  the  complete  disappearance  of  all  blue  color.  The  end- 
point  here,  however,  is  very  sharply  defined,  contrary  to  the  condi- 
tions in  the  older  method. 

To  prevent  the  annoying  bumping  which  often  interferes  with 
the  titration,  a  medium-sized  piece  of  washed  absorbent  cotton4 
may  be  introduced  into  the  solution.  This  cotton  may  be  stirred 
about  through  the  solution  as  the  titration  proceeds  and  the  bump- 
ing thus  eliminated. 

Calculation. — Thirty  cubic  centimeters  of  Benedict's  solution 
is  completely  reduced  by  0.073  gram  of  dextrose.  If  y  represents 
the  number  of  cubic  centimeters  of  urine  necessary  to  reduce  the  30 
c.c.  of  the  solution  we  have  the  following  proportion  : 

y  :  0.073  : :  I0°  :  x  (percentage  of  dextrose). 

solution  are  the  same  as  those  already  described  in  connection  with  Benedict's 
qualitative  test,  see  p.  309.    The  third  solution  is  made  up  as  follows : 

Potassium  ferro-thiocyanate  solution  =  15  grams  of  potassium  ferrocyanide, 
62.5  grams  of  potassium  thiocyanate  and  50  grams  of  anhydrous  sodium  carbo- 
nate dissolved  in  water  and  made  up  to  500  c.c. 

These  three  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done  to 
prevent  deterioration. 

1  The  amount  added  depends  upon  the  dilution  to  which  the  solution  is  to  be 
subjected  in  titration.  For  this  reason  the  maximum  amount  of  sodium  car- 
bonate should  be  added  when  titrating  urines  containing  a  very  low  percentage 
of  sugar. 

2  Not  rapidly  enough,  however,  to  interfere  in  any  marked  degree  with  the 
continuous  vigorous  boiling  of  the  solution. 

3  The  exact  amount  to  run  in  depends  upon  the  intensity  of  the  remaining  blue 
color,  as  well  as  upon  the  sugar  content  of  the  urine.  The  10  drops  should  be 
added  at  one  time  only  when  urines  containing  a  very  low  percentage  of 

are  under  examination. 

4  Glass  wool  may  be  substituted  if  desired. 

25 


370  PHYSIOLOGICAL    CHEMISTRY. 

3.  Purdy's  Method. — Purdy's  solution1  is  a  modification  of 
Fehling's  solution  and  is  said  to  possess  greater  stability  than  the 
latter.  One  of  the  most  satisfactory  points  about  the  method  as 
suggested  by  Purdy  is  the  ease  with  which  the  exact  end-reaction 
may  be  determined.  In  determining  the  percentage  of  dextrose  by 
this  method  proceed  as  follows :  Place  35  c.c.  of  Purdy's  solution 
in  a  200  c.c.  Erlenmeyer  flask  and  dilute  the  fluid  with  approxi- 
mately two  volumes  of  distilled  water.  Fit  a  cork,  provided  with 
two  perforations,  to  the  neck  of  the  flask  and  through  one  perfora- 
tion introduce  the  tip  of  a  burette  and  through  the  second  perfora- 
tion introduce  a  tube  bent  at  right  angles  in  such  a  manner  as  to 
allow  the  steam  to  escape  and  keep  the  fumes  of  ammonia  away 
from  the  face  of  the  operator  as  completely  as  possible.2  Now 
bring  the  solution  to  the  boiling-point  and  add  the  urine,  drop  by 
drop,  until  the  intensity  of  the  blue  color  begins  to  diminish.  When 
this  point  is  reached  add  the  urine  somewhat  more  slowly  until 
the  blue  color  is  entirely  dissipated  and  an  absolutely  decolorised 
solution  remains.  Take  the  burette  reading  and  calculate  the  per- 
centage of  dextrose  in  the  urine  examined  according  to  the  method 
given  on  p.  371. 

Care  should  be  taken  not  to  boil  the  solution  for  too  long  a  per- 
iod, since,  under  these  conditions,  sufficient  ammonia  might  be 
lost  to  allow  the  cuprous  hydroxide  to  precipitate. 

Some  investigators  consider  it  to  be  advisable  to  dilute  the  urine 
before  applying  the  above  manipulation,  but  ordinarily  this  is  not 
necessary  unless  the  urine  has  a  high  content  of  dextrose   (5  per 

1  Purdy's  solution  has  the  following  composition  : 

Cupric  sulphate 4-752  grams. 

Potassium  hydroxide    23.5  grams. 

Ammonia  (U.  S.  P.,  sp.  gr.  0.9) 350.0  c.c. 

Glycerol  38.0  c.c. 

Distilled  water,  to  make  total  volume  1  liter. 

In  preparing  the  solution  bring  the  cupric  sulphate  and  potassium  hydroxide 
into  solution  in  separate  vessels,  mix  the  two  solutions,  cool  the  mixture  and 
add  the  ammonia  and  glycerol.  After  this  has  been  done  the  total  volume 
should  be  made  up  to  1  liter  with  distilled  water. 

Thirty-five  cubic  centimeters  of  Purdy's  solution  is  exactly  reduced  by  0.02 
gram  of  dextrose. 

2  This  side  tube  may  also  be  equipped  with  a  simple  air-valve,  thus  insuring  the 
exclusion  of  air  and  thereby  contributing  to  the  accuracy  of  the  determination, 
inasmuch  as  the  cuprous  salts  would  be  reoxidized  upon  coming  in  contact  with 
the  air.  If  one  is  careful  to  maintain  the  solution  continuously  at  the  boiling- 
point  throughout  the  entire  process,  however,  there  is  no  opportunity  for  air  to 
enter  and  therefore  no  need  of  an  air-valve. 


urine:  quantitative  analysis.  371 

cent  or  over).  In  this  event  the  urine  ma)  be  diluted  with  _*  3 
volumes  of  water  and  the  proper  correction  made  in  the  calculation. 
Calculation. — Thirty-five  e.e.  of  I'nrdv's  solution  is  completely 
reduced  by  0.02  gram  of  dextrose.  If  y  represents  the  number  of 
cubic  centimeters  of  undiluted  urine  necessary  to  reduce  35  c.c.  of 
Purdy's  solution,   we   have   the    following  proportion: 

y  :  0.02  : :  100 \x  (percentage  of  dextrose). 

4.  Fermentation  Method. — This  method  consists  in  the  meas 
urement  of  the  volume  of  carbon  dioxide  evolved  when  the  dex- 
trose of  the  urine  undergoes  fermentation  with  yeast.  None  of  the 
various  methods  whose  manipulation  is  based  upon  this  principle- 
is  absolutely  accurate.  The  method  in  which  Einhorn's  saccharo- 
meter (Fig.  2,  page  31)  is  the  apparatus  employed  is  perhaps  as 
satisfactory  as  any  for  clinical  purposes.  The  procedure  is  as 
follows:  Place  about  15  c.c.  of  urine  in  a  mortar,  add  about  1 
gram  of  yeast  (%6  of  the  ordinary  cake  of  compressed  yeast)  and 
carefully  crush  the  latter  by  means  of  a  pestle.  Transfer  the  mix- 
ture to  the  saccharometer,  being-  careful  to  note  that  the  graduated 
tube  is  completely  filled  and  that  no  air  bubbles  gather  at  the  top. 
Allow7  the  apparatus  to  stand  in  a  warm  place  (30  C.)  for  12  hours 
and  observe  the  percentage  of  dextrose  as  indicated  by  the  grad- 
uated scale  of  the  instrument.  Both  the  percentage  of  dextrose  and 
the  number  of  cubic  centimeters  of  carbon  dioxide  are  indicated 
by  the  graduations  on  the  side  of  the  saccharometer  tube. 

5.  Polariscopic  Examination. — Before  subjecting  urine  to  a 
polariscopic  examination  the  slightly  acid  fluid  should  be  decoli  ir- 
ized  as  thoroughly  as  possible  by  the  addition  of  a  little  plumbic 
acetate.  The  urine  should  be  well  stirred  and  then  filtered  through 
a  filter  paper  which  has  not  been  previously  moistened.  In  this 
way  a  perfectly  clear  and  almost  colorless  liquid  is  obtained. 

In  determining  dextrose  by  means  of  the  polariscope  it  should  be 
borne  in  mind  that  this  carbohydrate  is  often  accompanied  by  other 
optically  active  substances,  such  as  proteins,  laevulose,  /8-oxybutyric 
acid  and  conjugate  glycuronates  which  may  introduce  an  error  into 
the  polariscopic  reading ;  the  method  is,  however,  sufficiently  accur- 
ate for  practical  purposes. 

For  directions  as  to  the  manipulation  of  the  polariscope  see  page 

32- 


372  PHYSIOLOGICAL    CHEMISTRY. 

III.     Uric  Acid. 

i.  Folin-Shaffer  Method. — Introduce  ioo  c.c.1  of  urine  into  a 
beaker,  add  25  c.c.  of  the  Folin-Shaffer  reagent2  and  allow  the  mix- 
ture to  stand,3  without  further  stirring,  until  the  precipitate  has 
settled  (5-10  minutes).  Filter,  transfer  100  c.c.  of  the  nitrate 
to  a  200  c.c.  beaker  or  Erlenmeyer  flask,  add  5  c.c.  of  concentrated 
ammonia  and  allow  the  mixture  to  stand  for  24  hours.  Transfer 
the  precipitated  ammonium  urate  quantitatively  to  a  filter  paper,4 
using  10  per  cent  ammonium  sulphate  to  remove  the  final  traces 
of  the  urate  from  the  beaker.  Wash  the  precipitate  approximately 
free  from  chlorides  by  means  of  10  per  cent  ammonium  sulphate 
solution,5  remove  the  paper  from  the  funnel,  open  it  and  by  means 
of  hot  water  rinse  the  precipitate  back  into  the  beaker  in  which 
the  urate  was  originally  precipitated.  The  volume  of  fluid  at  this 
point  should  be  about  100  c.c.  Cool  the  solution  to  room  temper- 
ature, add  15  c.c.  of  concentrated  sulphuric  acid  and  titrate  at  once 
with  -%-q  potassium  permanganate,  K2Mn2Os,  solution.  The  first 
tinge  of  pink  color  which  extends  throughout  the  fluid  after  the  ad- 
dition of  tzvo  drops  of  the  permanganate  solution,  while  stirring 
with  a  glass  rod,  should  be  taken  as  the  end-reaction.  Take  the 
burette  reading  and  compute  the  percentage  of  uric  acid  present  in 
the  urine  under  examination. 

Calculation. — Each  cubic  centimeter  of  ■$-$  potassium  permangan- 
ate solution  is  equivalent  to  3.75  milligrams  (0.00375  gram)  of 
uric  acid.  The  100  c.c.  from  which  the  ammonium  urate  was  pre- 
cipitated is  equivalent  to  only  four-fifths  of  the  100  c.c.  of  urine 
originally  taken,  therefore  we  must  take  five-fourths  of  the  bur- 
ette reading  in  order  to  ascertain  the  number  of  cubic  centimeters 
of  the  permanganate  solution  required  to  titrate  100  c.c.  of  the 
original  urine  to  the  correct  end-point.  If  3;  represents  the  num- 
ber of  cubic  centimeters  of  the  permanganate  solution  required,  we 
may  make  the  following  calculation : 

y  X  0.00375  =  weight  of  uric  acid  in  100  c.c.  of  urine. 

1  It  is  preferable  to  use  more  than  100  c.c.  of  urine  if  the  fluid  has  a  specific 
gravity  less  than  1.020. 

2  The  Folin-Shaffer  reagent  consists  of  500  grams  of  ammonium  sulphate,  5 
grams  of  uranium  acetate  and  60  c.c.  of  10  per  cent  acetic  acid  in  650  c.c.  of 
distilled  wafer. 

3  The  mixture  should  not  be  allowed  to  stand  for  too  long  a  time  at  this  point, 
since  uric  acid  may  be  lost  through  precipitation. 

*The  Schleicher  and  Schiill  hardened  papers  or  the  Baker  and  Adamson 
washed,  ashless  variety  are  very  satisfactory  for  this  purpose. 

u  This  washing  may  be  conveniently  done  by  decantation  if  desired,  thus  retain- 
ing the  major  portion  of  the  precipitate  in  the  beaker  or  flask. 


urine:  quantitative  analysis. 

Because  of  the  solubility  of  the  ammonium  urate  a  correction  of 
3  milligrams  should  1>c  added  to  the  final  result. 

Calculate  the  quantity  of  uric  acid  in  the  twenty-four  hour  urine 
specimen. 

2.  Heintz  Method. — This  is  a  very  simple  method  and  was  the 
first  one  in  general  use  for  the  quantitative  determination  of  uric 
acid.  It  is  believed  to  be  somewhat  less  accurate  than  the  method 
just  described.  The  procedure  is  as  follows:  Place  [00  c.c.  of 
filtered  urine  in  a  beaker,  add  5  c.c.  of  concentrated  hydrochloric 
acid,  stir  the  fluid  thoroughly  and  stand  it  away  in  a  cool  place  for 
24  hours.  Filter  off  the  uric  acid  crystals  upon  a  washed,  dried  and 
weighed  filter  paper  and  wash  them  with  cold  distilled  water,  a  few- 
cubic  centimeters  at  a  time  until  the  chlorides  are  removed.  Now 
wash,  in  turn,  with  alcohol  and  with  ether  and  finally  dry  the 
paper  and  crystals  to  constant  weight  at  no  C.  In  the  process 
of  Avashing  the  uric  acid  free  from  chlorides  an  error  is  introduced, 
since  every  cubic  centimeter  of  water  so  used  dissolves  0.00004 
gram  of  uric  acid.  For  this  reason  a  correction  is  necessary.  Jt 
has  been  suggested  that  the  pigment  of  the  crystals  is  equivalent 
in  weight  to  the  amount  of  uric  acid  dissolved  by  the  first  30  c.c. 
of  water,  and  this  factor  should  be  taken  into  account  in  the 
computation  of  the  percentage  of  uric  acid. 

Calculation. — Since  100  c.c.  of  urine  was  used  the  corrected 
weight  of  the  uric  acid  crystals,  in  grams,  will  express  the  percent- 
age of  uric  acid  present. 

3.  Kriiger  and  Schmidt's  Method. — This  method  serves  for 
the  detection  of  both  uric  acid  and  the  purine  bases.  The  principle 
involved  is  the  precipitation  of  both  the  uric  acid  and  the  purine 
bases  in  combination  with  copper  oxide  and  the  subsequent  decom- 
position of  this  precipitate  by  means  of  sodium  sulphide.  The 
uric  acid  is  then  precipitated  by  means  of  hydrochloric  acid  and 
the  purine  bases  are  separated  from  the  filtrate  in  the  form  of 
their  copper  or  silver  compounds.  The  nitrogen  content  of  the  pre- 
cipitates of  uric  acid  and  purine  bases  is  then  determined  by  means 
of  the  Kjeldahl  method  (see  p.  381)  and  the  corresponding  values 
for  uric  acid  and  purine  bases  calculated.  The  method  i^  as  fol- 
lows:   To  400  c.c.  of  albumin-free  urine1   in  a  liter  flask,2  add  24 

1  If  albumin  is  present,  the  urine  should  be  heated  10  boiling,  acidified  with 
acetic    acid    and    filtered. 

2  The  total  volume  of  urine  for  the  twenty-four  hours  should  be  sufficiently 

diluted  with  water  to  make  the   total   volume  "f   the   solution    1000  2000  CC. 


374  PHYSIOLOGICAL    CHEMISTRY. 

grams  of  sodium  acetate,  40  c.c.  of  a  solution  of  sodium  bisulphite1 
and  heat  the  mixture  to  boiling.  Add  40-80  c.c.2  of  a  10  per  cent 
solution  of  cupric  sulphate  and  maintain  the  temperature  of  the 
mixture  at  the  boiling-point  for  at  least  three  minutes.  Filter 
off  the  flocculent  precipitate,  wash  it  with  hot  water  until  the  wash 
water  is  colorless,  and  return  the  washed  precipitate  to  the  flask 
by  puncturing  the  tip  of  the  filter  paper  and  washing  the  precipi- 
tate through  by  means  of  hot  water.  Add  water  until  the  volume 
in  the  flask  is  approximately  200  c.c,  heat  the  mixture  to  boil- 
ing and  decompose  the  precipitate  of  copper  oxide  by  the  addition  of 
30  c.c.  of  sodium  sulphide  solution.3  After  decomposition  is  com- 
plete, the  mixture  should  be  acidified  with  acetic  acid  and  heated 
to  boiling  until  the  separating  sulphur  collects  in  a  mass.  Filter 
the  hot  fluid  by  means  of  a  filter  pump,  wash  with  hot  water,  add 
10  c.c.  of  10  per  cent  hydrochloric  acid  and  evaporate  the  filtrate  in 
a  porcelain  dish  until  the  total  volume  has  been  reduced  to  about  ten 
cubic  centimeters.  Permit  this  residue  to  stand  about  two  hours 
to  allow  for  the  separation  of  the  uric  acid,,  leaving  the  purine  bases 
in  solution.  Filter  off  the  precipitate  of  uric  acid,  using  a  small 
filter  paper,  and  wash  the  uric  acid,  with  water  made  acid  with 
sulphuric  acid,  until  the  total  volume  of  the  original  filtrate  and  the 
wash  water  aggregates  75  c.c.  Determine  the  nitrogen  content  of 
the  precipitate  by  means  of  the  Kjeldahl  method  (see  p.  381)  and 
calculate  the  uric  acid  equivalent. 

Calculation. — In  calculating  the  uric  acid  value  from  the  total 
nitrogen  simply  multiply  the  latter  by  three  and  add  0.0035  to  the 
product  as  a  correction  for  the  uric  acid  remaining  in  solution  in 
the  75  c.c. 

IV.     Urea. 

1.  Knop-Hiifner  Hypobromite  Method  (using  Marshall's 
Urea  Apparatus). — Place  the  thumb  over  the  side  opening  of 
the  bulbed-tube  of  the  apparatus  (Fig.  118,  p.  375)  and  carefully 

1  A  solution  containing  50  grams  of  Kahlbaum's  commercial  sodium  bisulphite 
in  100  c.c.  of  water. 

2  The  exact  amount  depending  upon  the  content  of  the  purine  bases. 

3  This  is  made  by  saturating  a  one  per  cent  solution  of  sodium  hydroxide  with 
hydrogen  sulphide  gas  and  adding  an  equal  volume  of  one  per  cent  sodium 
hydroxide. 

Ordinarily  the  addition  of  30  c.c.  of  this  solution  is  sufficient,  but  the  presence 
of  an  excess  of  sulphide  should  be  proven  by  adding  a  drop  of  lead  acetate  to  a 
drop  of  the  solution.  Under  these  conditions  a  dark  brown  color  will  show  the 
presence  of  an  excess  of  sodium  sulphide. 


urine:  quantitative  analv-i-. 


375 


Fig.   ■  r8. 


till  i lie  tube  with  sodium  hypobromite  solution.1  Close  the  opening 
in  the  end  of  the  tube  with  a  rubber  stopper,  incline  the  tube  to 
allow  air-bubbles  to  escape  and  finally  invert  the  tube  and  fix  the 
stoppered  end  in  the  saucer  shaped  vessel.  l'.\  means  of  the  grab" 
uated  pipette  rapidly  introduce  i  c.c. 
of  urine-  into  the  hypobromite  solution 
through  the  side  opening  of  the  bulbed- 
tube.  Withdraw  the  pipette  immedi- 
ately after  the  urine  has  been  intro- 
duced. When  the  decomposition  oi 
the  urea  is  completed  (10-20  minutes) 
gently  tap  the  bulbed-tube  with  the 
finger  in  order  to  dislodge  any  gas 
bubbles  which  may  have  collected  on 
the  inner  surface  of  the  glass.  The 
atmospheric  pressure  should  now  be 
equalized  by  attaching  the  funnel-tube 
to  the  bulbed-tube  at  the  side  opening 
and  introducing  hypobromite  solution 
into  it  until  the  columns  of  liquid  in 
the  two  tubes  are  uniform  in  height. 
The  graduated  scale  of  the  bulbed- 
tube  should  now  be  read  in  order 
to  determine  the  number  of  cubic 
centimeters  of  nitrogen  gas  evolved. 
By  means  of  the  appended  formula 
the  weight  of  the  urea  present  in  the  urine  under  examination  may 
be  computed. 

Calculation/' — By  properly  substituting  in  the  following  formula 
the  weight  of  urea,  in  grams,  contained  in  the  volume  of  urine  de- 
composed  ( 1  c.c.  or  more )   may  readily  be  determined  : 

1  The  ingredients  of  the  sodium  hypobromite  solution  should  be  prepared  in  the 
form  of  tzvo  separate  solutions.  When  needed  for  use  mix  equal  volumes  of 
solution  a,  solution  b  and  water. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams  of  bromine 
and  make  the  total  volume  of  the  solution   i  liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of  I.250.  This 
is  approximately  a  22.5  per  cenl    solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles. 

"Ordinarily  1  c.c.  of  urine  is  sufficient;  more  may  be  used,  however,  if  it > 
content  of  urea  is  very  low. 

3 0.003665  =  coefficient  of  expansion  of  gases  for  i°  C.  354.5  =  number  of  cc 
of  nitrogen  gas  evolved  from  1  gram  of  urea. 


'■  Hl'lll'll'l    "■   '      ■  ■        ' 

Marshall's  Urea  Apparatus. 
(Tyson.) 

a,  Bulbed  measuring  tube ;  b, 
saucer-shaped  vessel ;  c,  graduated 
pipette  ;  d,  funnel-tube. 


3^6  PHYSIOLOGICAL    CHEMISTRY. 

354.5  X  760(1  +0.003665O 
w  =  weight  of  urea,  in  grams. 

v  =  observed  volume  of  nitrogen  expressed  in  cubic  centimeters. 
p  —  barometric  pressure  expressed  in  mm.  of  mercury. 
T  =  tension  of  aqueous  vapor1  for  temperature  t. 
t  =  temperature  (centigrade). 

If  we  wish  to  calculate  the  percentage  of  urea  we  may  do  so  by 
means  of  the  following  proportion  in  which  y  represents  the  vol- 
ume of  urine  used  and  w  denotes  the  weight  of  the  urea  contained 
in  the  volume  y : 

y  :  w : :  100 :  x   (percentage  of  urea) . 

Sodium  hypobromite  solution  may  also  be  employed  for  the  de- 
termination of  urea  in  the  apparatus  devised  by  Htifner  which  is 
pictured  in  Fig.  119,  page  377. 

2.  Knop-Hiifner  Hypobromite  Method  (using  the  Doremus- 
Hinds  Ureometer). — In  common  with  the  method  already  de- 
scribed this  method  depends  upon  the  measurement  of  the  volume 
of  nitrogen  gas  liberated  when  the  urea  of  the  urine  is  decomposed 
by  means  of  sodium  hypobromite  solution.  The  Doremus-Hinds 
ureometer  (Fig.  120,  p.  378),  is  one  of  the  simplest  and  cheapest 
forms  of  apparatus  in  general  use  for  the  determination  of  urea  by 
the  hypobromite  process.  In  using  this  apparatus  proceed  as  fol- 
lows :  Fill  the  side  tube  B  and  the  lumen  of  the  stopcock  C  with  the 
urine  under  examination.  Carefully  wash  out  tube  A  with  water 
and  introduce  into  it  sodium  hypobromite  solution2  being  careful  to 
fill  the  bulb  sufficiently  full  to  prevent  the  entrance  of  air  into  the 

1  The  values  of  T  for  the  temperatures  ordinarily  met  with  are  given  in  the 
following  table : 

Tension  in  Tension  in 

Temp.  Mm.  Temp.  Mm. 

15°  C... 12.677  210  C 18.505 

160  C 13.519  22°  C 19.675 

17°  C 14.009  23°  C 20.909 

180  C 15-351  24°  C 22.211 

19°  C 16.345  25°  C 23.582 

20°    C 17.396 

2  For  directions  as  to  the  preparation  of  this  solution  see  page  375. 


urine:  quantitative  analysis. 


graduated    portion.      Now    allow    I  '  ";-  "9« 

c.c.  of  urine1   to  flow  from  tube  B 

into  tube  A  and  after  the  evolution 

of  gas  bubbles  has  ceased    (10-20 

minutes)    take   the   reading'   of   the 

graduated  scale  on  tube  A. 

In  common  with  all  other  meth- 
ods which  are  based  upon  the  de- 
composition of  urea  by  means  of 
hypobromite  solution,  this  method 
is  not  absolutely  correct.  It  is. 
however,  sufficiently  accurate  for 
ordinary  clinical  purposes. 

Calculation. — Observe  the  reading 
on  the  graduated  scale  of  tube  A. 
This  tube  is  so  graduated  as  to  rep- 
resent the  weight  of  urea,  in  grams, 
per  cubic  centimeter  of  urine.  If  we 
wish  to  compute  the  percentage  of 
urea  present  this  may  be  done  very 
readily  by  simply  moving  the  deci- 
mal point  two  places  to  the  right,  e. 
g.,  if  the  reading  is  0.02  gram  the 
urine  contains  2  per  cent  of  urea. 

3.  Folin's  Method. — This  is  one 
of  the  most  accurate  methods  yet 
devised  for  the  determination  of 
urea  in  the  urine.     The  procedure 

is  as  follows :  Place  5  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer 
flask  and  add  to  it  5  c.c.  of  concentrated  hydrochloric  acid.  20  grams 
of  crystallized  magnesium  chloride,  a  piece  of  paraffin  the  size  of  a 
hazel  nut  and  2-3  drops  of  a  1  per  cent  aqueous  solution  of  "aliz- 
arin red.''  Insert  a  Folin  safety  tube  I  Fig.  1  _•  i .  p.  370")  into  the 
neck  of  the  flask  and  boil  the  mixture  until  each  drop  of  reflow  from 
the  safety  tube  produces  a  very  perceptible  bump:  the  heat  is  then 
reduced  somewhat  and  continued  one  and  one-half  hours.  The 
contents  of  the  flask  must  not  remain  alkaline  and  to  obviate  this, 
at  the  first  appearance  of    a  reddish   tinge  in  the  contents  <>\   the 

'If  the  content  of  urea  in  the  urine  under  examination  is  large,  the  urine 
may  be  diluted  with  water  before  determining  the  urea.  If  this  is  done  it  must 
of  course  be  taken  into  consideration  in  computing  the  content   of  urea. 


Hufner's  Urea  Apparatus. 


378 


PHYSIOLOGICAL    CHEMISTRY. 


flask  a  few  drops  of  the  acid  distillate  are  shaken  back  into  the  flask. 
At  the  end  of  1^2  hours  the  contents  of  the  vessel  are  transferred  to 
a  1  liter  flask  with  about  700  c.c.  of  distilled  water,  about  20  c.c.  of 
10  per  cent  potassium  hydroxide  or  sodium  hydroxide  solution  is 
added  and  the  mixture  distilled  into  a  known  volume  of  -^L  sulphuric 
acid  until  the  contents  of  the  flask  are  nearly  dry  or  until  the  dis- 


FlG. 


tillate  fails  to  give  an  alkaline  reaction 


usmp' 


to  litmus,  showing  the  absence  of 
ammonia.  The  time  devoted  to  this 
process  is  ordinarily  about  an  hour. 
Boil  the  distillate  a  few  moments  to 
free  it  from  C02,  then  cool  and  titrate 
the  mixture  with  -j-q  sodium  hydroxide, 
"  alizarin  red  "  as  indicator. 
A  "  check "  experiment  should  al- 
ways be  made  to  determine  the  orig- 
inal ammonia  content  of  the  urine  and 
of  the  magnesium  chloride,  if  it  is  not 
absolutely  pure,  which  of  course  should 
be  subtracted  from  the  total  amount  of 
ammonia  as  determined  by  the  above 
process. 

The  Folin  method  is  extremely  accu- 
rate under  all  conditions  except  when 
the  urine  contains  sugar.     When  this 
^g^  is  the  case  the  carbohydrate  and  the 
urea    unite,    upon    being    heated,    and 
form  a  very  stable  combination.     For 
this   reason   the   Folin   method   is   not 
suitable  for  use  in  the  examination  of 
such  urines.     The  best  method  for  use  under  such  conditions  is  the 
combination  Morner-Sjoqvist-Folin  method  which  is  given  below. 

4.  Morner-Sjoqvist-Folin  Method. — As  has  already  been  stated 
in  the  last  experiment  this  method  excels  the  Folin  method  in  ac- 
curacy only  in  the  determination  of  urea  in  the  presence  of  carbo- 
hydrate bodies.  Briefly  the  procedure  is  as  follows  ■}  Bring  the 
major  portion  of  1.5  gram  of  powdered  barium  hydroxide  into 
solution  in  5  c.c.  of  urine  in  a  small  flask,  and  treat  the  mixture 
with  100  c.c.  of  an  alcohol-ether  solution,  consisting  of  two  volumes 

1The  original  description  of  the  method  may  be  found  in  an  article  by  Morner: 
Skandinavisches  Archiv  fur  Physiologic,  1903,  xiv,  p.  297. 


DOREMUS-HlNDS    UrEOMETER. 


urine:  quantitative  analysis. 


379 


Fig. 


of  97  per  cent  alcohol  and  one  volume  of  ether.  Stopper  the  flask 
and  allow  it  to  stand  12  24  hours.  Filter  off  the  precipitate,  wash  it 
with  the  alcohol-ether  mixture  and  remove  the  alcohol  and  ether 
from  the  filtrate  by  distillation,  being  careful  to  keep  the  tempera- 
ture of  the  mixture  below  50°  C.1  Treat  the  remaining  fluid  1  aboul 
25  c.c.)  with  2  c.c.  of  hydrochloric  acid  (sp.  gr.  [.124)  transfer  it 
carefully  to  a  200  c.c.  flask  and  evaporate  the  mixture  to  drynes 
a  water-bath.  Now  add  20  grams  of 
crystallized  magnesium  chloride  and  2 
c.c.  of  concentrated  hydrochloric  acid 
to  the  residue  and  after  fitting  the  flask 
with  a  return  cooler  boil  the  mixture 
on  a  wire  gauze  over  a  small  flame  for 
two  hours.  Cool  the  solution,  dilute 
to  750  c.c.  or  to  1000  c.c.  with  water, 
render  the  mixture  alkaline  with  potas- 
sium hydroxide  or  sodium  hydroxide, 
distil  off  the  ammonia  and  collect  it 
in  an  acid  solution  of  known  strength. 
Boil  the  distillate  to  remove  carbon 
dioxide,  cool  and  titrate  with  an  alkali 
of  known  strength.  In  this  method, 
as  well  as  in  Folin's  method  (see  p. 
377),  correction  must  be  made  for  the 
ammonia  originally  present  in  the 
urine  and  in  the  magnesium  chloride. 

5.  Benedict  and  Gephart's  Method. 
— Introduce  into  a  rather  wide  test- 
tube  or  a  small  Erlenmeyer  flask  5 
c.c.  of  the  urine  under  examination 
and  an  equal  volume  of  dilute  (1:4) 
hydrochloric  acid.      Cover  the   mouth 

of  the  tube  or  flask  with  a  cap  made  by  folding  a  piece  of  lead-foil 
over  the  top  and  place  the  vessel  in  an  autoclave  maintained  at  a 
temperature  of  150-155°  C.  for  one  and  one-half  hours.'-  After  the 
autoclave  has  cooled,  wash  the  contents  of  the  tube  into  an  Soo  c.c. 
Kjeldahl  distillation  flask,  dilute  the  urine  mixture  to  about  400  c.c. 
with  distilled  water,  add  20  c.c.  of  a  to  per  cent  solution  of  sodium 

1  There  is  some  decomposition  of  urea  at  6o°  C. 

"This  corresponds  to  a  pressure  of  about  six  kilograms  per  square  centimeter. 

The  cap?  may  be  conveniently  labelled  with  a  stylus. 


Folin's  Urea  Appaiu 


38o 


PHYSIOLOGICAL    CHEMISTRY. 


hydroxide  and  distil  about  40  minutes  into  an  excess  of  standard 
acid.  Complete  the  process  as  described  under  the  Kjeldahl  method 
(p.  381).  Subtract  the  original  ammonia  content  of  the  urine  from 
the  total  ammonia  content  (ammonia  -+-  urea)  as  determined  by  the 
Kjeldahl  method. 

V.  Ammonia. 

1.  Folin's  Method. — Place  25  c.c.  of  urine  in  an  aerometer  cylin- 
der, 30-40  cm.  in  height  (Fig.  122,  below),  add  about  one  gram  of 
dry  sodium  carbonate  and  introduce  some  crude  petroleum  to  pre- 
vent foaming.  Insert  into  the  neck  of  the  cylinder  a  rubber  stopper 
provided  with  two  perforations  into  each  of  which  passes  a  glass 
tube  one  of  which  reaches  below  the  surface  of  the  liquid.  The 
shorter  tube  (10  cm.  in  length)  is  connected  with  a  calcium  chloride 
tube  filled  with  cotton  and  this  tube  is  in  turn  joined  to  a  glass  tube 
extending  to  the  bottom  of  a  500  c.c.  wide-mouthed  flask  which  is 
intended  to  absorb  the  ammonia  and  for  this  purpose  should  con- 
tain 20  c.c.  of  f-Q  sulphuric  acid,  200  c.c.  of  distilled  water  and  a  few 
drops  of  an  indicator  ("alizarin  red").  To  insure  the  complete 
absorption  of  the  ammonia  the  absorption  flask  is  provided  with  a 

Fig.  122. 


Folin's  Ammonia  Apparatus. 


Folin  improved  absorption  tube  (Fig.  123,  p.  381)  which  is  very 
effective  in  causing  the  air  passing  from  the  cylinder  to  come  into 
intimate  contact  with  the  acid  in  the  absorption  flask.  In  order  to 
exclude  any  error  due  to  the  presence  of  ammonia  in  the  air  a  similar 


urine:  quantitative  anal 


38! 


Fig.   1 .-  •;. 


absorption  apparatus  to  the  one  just  described  is  attached  to  the 
other  side  of  the  aerometer  cylinder,  thus  insuring  the  passaj 
ammonia-free  air   into  the   cylinder.     With   an 

ordinary  filter  pump  and  good  water  pressure  the 
last  trace  of  ammonia  should  he  removed  from 
the  cylinder  in  about  one  and  one-half  hours.1 

The  number  of  cubic  centimeters  of  the  ,N(I  sul- 
phuric acid  neutralized  by  the  ammonia  of  the 
urine  may  be  determined  by  direct  titration  with 
f^  sodium  hydroxide. 

This  is  one  of  the  most  satisfactory  methods 
yet  devised   for  the  determination   of  ammonia. 

Calculation. — Subtract  the  number  of  cubic 
centimeters  of  j-q  sodium  hydroxide  used  in  the 
titration  from  the  number  of  cubic  centimeters 
of  jq  sulphuric  acid  taken.  The  remainder  is 
the  number  of  cubic  centimeters  of  yo  sulphuric 
acid  neutralized  by  the  NH3  of  the  urine.  1  c.c. 
of  jQ  sulphuric  acid  is  equivalent  to  0.001/  gram 
of  NH3.  Therefore  if  y  represents  the  volume 
of  urine  used  in  the  determination  and  y'  the 
number  of  cubic  centimeters  of  j-$  sulphuric  acid  neutralized  b\  the 
NHZ  of  the  urine,  we  have  the  following  proportion  : 

y  :  100  ::y'  X  0.0017 :  x  (percentage  of  XH3  in  the  urine  examined). 


Folix  Improved  Ab- 
sorption Tube. 


Calculate  the  quantity  of  NH3  in  the  twenty-four  hour  urine 
specimen. 

VI.  Nitrogen. 

Kjeldahl  Method.2 — The  principle  of  this  method  is  the  con- 
version of  the  various  nitrogenous  bodies  of  the  urine  into  ammon- 
ium sulphate  by  boiling  with  concentrated  sulphuric  acid,  the  subse- 
quent decomposition  of  the  ammonium  sulphate  by  means  of  a  fixed 
alkali  (NaOH)  and  the  collection  of  the  liberated  ammonia  in  an 
acid  of  known  strength.      Finally,  this  partly  neutralized  acid  solu- 

1  With  any  given  filter  pump  a  "check"  test  should  be  made  with  urine  or 
better  with  a  solution  of  an  ammonium  salt  of  known  strength  to  determine  how 

long  the  air  current  must  be  maintained  to  remove  all  the  ammonia  from  25  c.c. 
of  the   solution. 

"There  are  numerous  modifications  of  the  original  Kjeldahl  method;  the  one 
described  here,  however,  has  given  excellent  satisfaction  and  is  recommended 
for  the  determination  of  the  nitrogen  content  of  urine. 


382  PHYSIOLOGICAL    CHEMISTRY. 

tion  is  titrated  with  an  alkali  of  known  strength  and  the  nitrogen 
content  of  the  urine  under  examination  computed. 

The  procedure  is  as  follows :  Place  5  c.c.  of  urine  in  a  500  c.c. 
long-necked,  Jena  glass  Kjeldahl  flask,  add  20  c.c.  of  concentrated 
sulphuric  acid  and  about  0.2  gram  of  cupric  sulphate  and  boil  the 
mixture  for  some  time  after  it  is  colorless  (about  one  hour) .  Allow 
the  flask  to  cool  and  dilute  the  contents  with  about  200  c.c.  of  water. 
Add  a  little  more  of  a  concentrated  solution  of  NaOH  than  is  nec- 
essary to  neutralize  the  sulphuric  acid1  and  introduce  into  the  flask 
a  little  coarse  pumice  stone  or  a  few  pieces  of  granulated  zinc,2  to 
prevent  bumping,  and  a  small  piece  of  paraffin  to  lessen  the  tendency 
to  froth.  By  means  of  a  safety-tube  connect  the  flask  with  a  con- 
denser so  arranged  that  the  delivery-tube  passes  into  a  vessel  con- 
taining a  known  volume  (the  volume  used  depending  upon  the  nitro- 
gen content  of  the  urine)  of  y^-  sulphuric  acid,  using  care  that  the 
end  of  the  delivery-tube  reaches  beneath  the  surface  of  the  fluid.3 
Mix  the  contents  of  the  distillation  flask  very  thoroughly  by  shak- 
ing and  distil  the  mixture  until  its  volume  has  diminished  about 
one-half.  Titrate  the  partly  neutralized  y^-  sulphuric  acid  solution 
by  means  of  yy  sodium  hydi oxide,  using  congo  red  as  indicator,  and 
calculate  the  content  of  nitrogen  of  the  urine  examined. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  yg- 
sodium  hydroxide  used  in  the  titration  from  the  number  of  cubic 
centimeters  of  yg-  sulphuric  acid  taken.  The  remainder  is  equiva- 
lent to  the  number  of  cubic  centimeters  of  jq  sulphuric  acid,  neu- 
tralised by  the  ammonia  of  the  urine.  One  c.c.  of  j-$  sulphuric  acid 
is  equivalent  to  0.0014  gram  of  nitrogen.  Therefore,  if  3'  repre- 
sents the  volume  of  urine  used  in  the  determination,  and  y'  the 
number  of  cubic  centimeters  of  yg-  sulphuric  acid  neutralised  by  the 
ammonia  of  the  urine,  we  have  the  following  proportion : 

y:  100:  :y'  X  0.0014  :.r   (percentage  of  nitrogen  in  the  urine  ex- 
amined). 

Calculate  the  quantity  of  nitrogen  in  the  twenty-four  hour  urine 
specimen. 

1  This  concentrated  sodium  hydroxide  solution  should  be  prepared  in  quantity 
and  "check"  tests  made  to  determine  the  volume  of  the  solution  necessary  to 
neutralize  the  volume  (20  c.c.)  of  concentrated  sulphuric  acid  used. 

2  Powdered  zinc  may  be  substituted. 

3  This  delivery-tube  should  be  of  large  caliber  in  order  to  avoid  the  "  sucking 
back"  of  the  fluid. 


urine:  quantitative  analysi  383 

VII.   Hippuric  Acid. 

Dakin's  Methods.1  Preliminary  Procedure.  Place  [50C.C.  (or 
more)  of  the  urine  under  examination  in  a  porcelain  evaporating 
dish  and  evaporate  almost  to  dryness  upon  a  water  hath.  \dd 
about  1  gram,  of  sodium  dihydrogen  phosphate,  about  25  grams  of 

gypsum  (CaSO,,  2H20)  and  rub  up  with  a  pestle  and  stir  with  a 
spatula  until  a  uniform  mixture  results.  Dry  the  powder  thus  pro 
duced  in  a  water-oven  for  about  two  hours,  at  the  end  of  which 
period  it  should  he  rubbed  up  a  second  time,  to  remove  lumps,  and 
transferred  to  a  Schleicher  and  Schtill  "extraction  shell"  and  ex- 
tracted in  a  Soxhlet  apparatus  in  the  usual  way.  (see  p.  410).  The 
extraction  medium  is  ethyl  acetate  and  the  flask  containing  the  acetate 
should  be  strongly  heated  over  a  sand-bath2  for  about  two  hours. 
The  ethyl  acetate  extract  is  now  transferred  to  a  separators-  funnel, 
and  the  original  flask  rinsed  with  sufficient  fresh  ethyl  acetate  to 
make  the  total  volume  in  the  separately  funnel3  about  100  c.c. 
Wash  the  ethyl  acetate  solution  five  limes  with  a  saturated  solution 
of  sodium  chloride,  using  8  c.c.  of  the  sodium  chloride  solution  at 
each  extraction,  shaking  vigorously  and  removing  the  sodium 
chloride  extract  in  each  case  before  adding  fresh  sodium  chloride 
solution.  The  sodium  chloride  removes  the  urea  completely  and  the 
hippuric  acid  is  then  determined  in  the  urea-free  solution  by  the  fol- 
lowing volumetric  or  gravimetric  procedure : 

1.  Volumetric  Determination. — Transfer  the  urea-free  ethyl 
acetate  solution,  prepared  as  described  above,  to  a  Kjeldahl  flask. 
add  about  25  c.c.  of  water,  a  small  piece  of  pumice  stone  to  prevent 
bumping,  attach  a  condenser  and  distil  off  the  ethyl  acetate4  over 
a  free  flame.  After  practically  all  of  the  ethyl  acetate  has  been  dis- 
tilled off  the  nitrogen  in  the  remaining  solution  should  be  deter- 
mined by  means  of  the  Kjeldahl  method  (see  p.  381). 

The  main  source  of  error  in  this  method  is  the  fact  that  any 
nitrogen  present  in  the  form  of  phenaceturic  acid  or  indole  aeetie 
acid  is  determined  as  hippuric  acid  nitrogen.  The  error  from  this 
source  is,  however,  usually  trifling. 

Calculation. — Calculate  as  usual  for  nitrogen  determinations,  re- 

1  Private  communication  to  the  author  from  Dr.  H.  D.  Dakin. 

2 A  water-bath   cannot   be   substituted   inasmuch    as   the    resultant    extr 
would  be  too  slow. 

3  This  ethyl  acetate  solution  contain-  hippuric  acid,  urea  and  other  substances. 

4 The  ethyl  acetate  after  separation  from  the  watery  layer  of  the  distillate 
may  be  dried  over  calcium  chloride  and  used  again. 


384  PHYSIOLOGICAL    CHEMISTRY. 

membering  that  1  c.c.  of  j-q  sulphuric  acid  is  equivalent  to  0.0179 
gram  hippuric  acid. 

2.  Gravimetric  Determination. — The  urea-fr.ee  ethyl  acetate  so- 
lution, contained  in  the  separatory  funnel,  after  washing  with 
sodium  chloride  solution,  as  described  under  Preliminary  Procedure, 
p.  383,  is  washed  with  5  c.c.  of  distilled  water  to  remove  the  major 
portion  of  the  sodium  chloride.  Transfer  the  solution  from  the 
separatory  funnel  to  a  round-bottomed  flask  and  subject  it  to  a  steam 
distillation  in  the  usual  way.  A  slow  current  of  steam  should  be 
used  while  the  ethyl  acetate  is  being  distilled  off  and  later  a  more 
rapid  current  may  be  employed.  The  distillation  should  be  con- 
tinued for  twenty  minutes.  Now  add  about  0.1  gram  of  charcoal 
to  the  aqueous  solution  which  is  heated  to  boiling  and  filtered  hot. 
Evaporate  the  solution  in  a  weighed  Jena  glass  dish  on  a  water-bath 
until  the  volume  of  the  solution  is  reduced  to  about  3  c.c.  Stand 
the  dish  in  a  warm  place  until  evaporation  is  complete  and  a  crys- 
talline residue  remains.  Wash  the  residue,  in  turn,  with  2  c.c.  of 
dry  ether,  and  1  c.c.  of  water,  dry  it  in  an  air-bath  at  ioo°  C.  and 
weigh.  If  it  is  so  desired  the  residue  may  be  recrystallized  from  a 
little  hot  water  and  the  melting-point  determined.  Pure  hippuric 
acid  melts  at  1870  C.  Contamination  with  phenaceturic  acid  may 
be  detected  both  by  the  melting-point  and  the  microscopical  char- 
acteristics. 

VIII.  Sulphur. 

1.  Total  Sulphates. — Folin's  Method. — Place  25  c.c.  of  urine 
in  a  200-250  c.c.  Erlenmeyer  flask,  add  20  c.c.  of  dilute  hydrochloric 
acid1  (1  volume  of  concentrated  HC1  to  4  volumes  of  water)  and 
gently  boil  the  mixture  for  20-30  minutes.  To  minimize  the  loss  of 
water  by  evaporation  the  mouth  of  the  flask  should  be  covered  with 
a  small  watch  glass  during  the  boiling  process.  Cool  the  flask  for 
2-3  minutes  in  running  water,  and  dilute  the  contents  to  about  150 
c.c.  by  means  of  cold  water.  Add  10  c.c.  of  a  5  per  cent  solution  of 
barium  chloride  slowly,  drop  by  drop,  to  the  cold  solution.2  The 
contents  of  the  flask  should  not  be  stirred  or  shaken  during  the  ad- 
dition of  the  barium  chloride.     Allow  the  mixture  to  stand  at  least 

1  If  it  is  desired,  50  c.c.  of  urine  and  4  c.c.  of  concentrated  acid  may  be  used 
instead. 

2  A  dropper  or  capillary  funnel  made  from  an  ordinary  calcium  chloride  tube 
and  so  constructed  as  to  deliver  10  c.c.  in  2-3  minutes  is  recommended  for  use  in 
adding  the  barium  chloride. 


urine:  quantitative  analysis.  J85 

one  hour,  then  shake  up  (he  solution  and  filter  it  through  a  weighed 
Gooch  crucible.1 

Wash  the  precipitate  of  I'.aSo,  with  aboul  250  c.c.  of  cold  water, 
dry  it  in  an  air-hath  or  over  a  very  low  flame,  then  ignite,2  cool  and 
weigh. 

Calculation. — Subtract  the  weight  of    the  Gooch  crucible   from 
the  weight  of  the  crucible  and  the   I  in  SO,  precipitate  n,  ..htain  the 
weight  of  the  precipitate.     The  weight  of  So,:;  in  the  volume  of 
urine  taken  may  be  determined  by  means  of  the  following  propor 
tion. 

Mol.  wt.  Wt.  of  Mo',  wt. 

BaS04:BaS04:  :SO:j:.r  (wt.  of  SOa  in  gram.). 
ppt. 

Representing  the  weight  of  the  BaS04  precipitate  by  y  and  substi- 
tuting the  proper  molecular  weights,  we  have  the  following  pro- 
portion : 

231.7:3/:  -.yg.^-.x  (wt.  of  S03  in  grams  in  the  quantity  of  urine 

used). 

Calculate  the  quantity  of  S03  in  the  twenty-four  hour  specimen 
of  urine. 

To  express  the  result  in  percentage  of  SOs  simply  divide  the  value 
of  x,  as  just  determined,  by  the  quantity  of  urine  used. 

2.  Inorganic    Sulphates. — Folin's   Method. — Place    25    c.c.    of 

1If  a  Gooch  crucible  is  not  available  the  precipitate  of  BaSO.  may  be  filtered 
off  upon  a  washed  filter  paper  (Schleicher  &  Schull's,  No.  589,  blue  ribbon)  and 
after  washing  the  precipitate  with  about  250  c.c.  of  cold  water  the  paper  and 
precipitate  may  be  dried  in  an  air-bath,  or  over  a  low  flame.  The  ignition  may 
then  be  carried  out  in  the  usual  way  in  the  ordinary  platinum  or  porcelain 
crucible.  In  this  case  correction  must  be  made  for  the  weight  of  the  ash  of  tin- 
filter  paper  used. 

2  Care  must  be  taken  in  the  ignition  of  precipitates  in  Gooch  crucibles.  The 
flame  should  never  be  applied  directly  to  the  perforated  bottom  or  to  the  sides 
of  the  crucible,  since  such  manipulation  is  invariably  attended  by  mechanical 
losses.  The  crucibles  should  always  be  provided  with  lids  and  tight  bottoms 
during  the  ignition.  In  case  porcelain  Gooch  crucibles,  whose  bottoms  are  nol 
provided  with  a  non-perforated  cap,  are  used,  the  crucible  may  be  placed  upon 
the  lid  of  an  ordinary  platinum  crucible  durin.n  ignition.  The  lid  should  be  sup- 
ported on  a  triangle,  the  crucible  placed  upon  the  lid  and  the  Same  applied  to 
the  improvised  bottom.  Ignition  should  be  complete  in  to  minutes  if  no  organic 
matter  is  present. 

3  It  is  considered  preferable  by  many  investigators  to  express  all  sulphur  values 
in  terms  of  S  rather  than  SO3. 

26 


386  PHYSIOLOGICAL    CHEMISTRY. 

urine  and  ioo  c.c.  of  water  in  a  200-250  c.c.  Erlenmeyer  flask  and 
acidify  the  diluted  urine  with  10  c.c.  of  dilute  hydrochloric  acid  ( 1 
volume  of  concentrated  HC1  to  4  volumes  of  water).  In  case  the 
urine  is  dilute  50  c.c.  may  be  used  instead  of  25  c.c.  and  the  volume 
of  water  reduced  proportionately.  Add  10  c.c.  of  5  per  cent  barium 
chloride  slowly,  drop  by  drop,  to  the  cold  solution  and  from  this 
point  proceed  as  indicated  in  the  method  for  the  determination  of 
Total  Sulphates,  page  384. 

Calculate  the  quantity  of  inorganic  sulphates,  expressed  as  S03,  in 
the  twenty-four  hour  urine  specimen. 

Calculation. — Calculate  according  to  the  directions  given  under 
Total  Sulphates,  page  385. 

3.  Ethereal  Sulphates. — Foliris  Method. — Place  125  c.c.  of 
urine  in  an  Erlenmeyer  flask  of  suitable  size,  dilute  it  with  75  c.c.  of 
water  and  acidify  the  mixture  with  30  c.c.  of  dilute  hydrochloric 
acid  (1  volume  of  concentrated  HO  to  4  volumes  of  water).  To 
the  cold  solution  add  20  c.c.  of  a  5  per  cent  solution  of  barium  chlor- 
ide, drop  by  drop.1  Allow  the  mixture  to  stand  about  one  hour,  then 
filter  it  through  a  dry  filter  paper.2  Collect  125  c.c.  of  the  filtrate 
and  boil  it  gently  for  at  least  one-half  hour.  Cool  the  solution, 
filter  off  the  precipitate  of  BaS04,  wash,  dry  and  ignite  it  according 
to  the  directions  given  on  page  385. 

Calculation. — The  weight  of  the  BaS04  precipitate  should  be 
multiplied  by  2  since  only  one-half  (125  c.c.)  of  the  total  volume 
(250  c.c.)  of  fluid  was  precipitated  by  the  barium  chloride.  The 
remaining  calculation  should  be  made  according  to  directions  given 
under  Total  Sulphates,  page  385. 

Calculate  the  quantity  of  ethereal  sulphates,  expressed  as  SOs,  in 
the  twenty- four  hour  urine  specimen. 

4.  Total  Sulphur. — Osborne-Folin  Method. — Place  25  c.c.  of 
urine3  in  a  200-250  c.c.  nickel  crucible  and  add  about  3  grams  of 
sodium  peroxide.  Evaporate  the  mixture  to  a  syrup  upon  a  steam 
water-bath  and  heat  it  carefully  over  an  alcohol  flame  until  it  solidi- 
fies (15  minutes).     Now  remove  the  crucible  from  the  flame  and 

1  See  note  (2)   at  the  bottom  of  page  384. 

2  This  precipitate  consists  of  the  inorganic  sulphates.  If  it  is  desired,  this 
BaSOi  precipitate  may  be  collected  in  a  Gooch  crucible  or  on  an  ordinary  quanti- 
tative filter  paper  and  a  determination  of  inorganic  sulphates  made,  using  the 
same  technique  as  that  suggested  on  p.  385.  In  this  way  we  are  enabled  to 
determine  the  inorganic  and  ethereal  sulphates  in  the  same  sample  of  urine. 

3  If  the  urine  is  very  dilute  50  c.c.  may  be  used. 


urine:  quantitative  analysi 

allow  it  to  cool.  Moisten  the  residue  with  i  2  c.c.  of  water,1 
sprinkle  about  7-8  grams  of  sodium  peroxide  over  the  contents  oi 
the  crucible  and  fuse  the  mass  over  an  alcohol  flame  for  about  io 
minutes.  Allow  the  crucible  to  cool  for  a  few  minutes,  add  about 
100  c.c.  of  water  to  the  contents  and  heal  at  leasl  one  half  hour 
over  an  alcohol  flame,  to  dissolve  the  alkali  and  decompose  the 
sodium  peroxide.  Next  rinse  the  mixture  into  a  400  1.50  c.c.  Er 
lenmeyer  flask,  by  means  of  hot  water,  and  dilute  it  to  about  25OC.C 
Heat  the  solution  nearly  to  the  boiling  point  and  add  concentrated 
hydrochloric  acid  slowly  until  the  nickelic  oxide,  derived  from  tin- 
crucible,  is  just  brought  into  solution.-  A  few  minutes  boiling 
should  now  yield  a  clear  s<  ilutii  in.  In  ease  t<  i<  1  lilt  le  pen ixide  or  too 
much  water  was  added  for  the  final  fusion  a  clear  solution  will  not 
be  obtained.  In  this  event  cool  the  solution  and  remove  the  in- 
soluble matter  by  filtration. 

To  the  clear  solution  add  5  c.c.  of  very  dilute  alcohol  1  about  [8 
20  per  cent)  and  continue  the  boiling  for  a  few  minutes.  The  alc<  >- 
hoi  is  added  to  remove  the  chlorine  which  was  formed  when  the 
solution  was  acidified.  Add  10  c.c.  of  a  10  per  cent  solution  of 
barium  chloride,  slowly,  drop  by  drop,3  to  the  liquid.  Allow  the 
precipitated  solution  to  stand  in  the  cold  two  days  and  then  filter  and 
continue  the  manipulation  according  to  the  directions  given  under 
Total  Sulphates,  page  384. 

Calculation. — Make  the  calculation  according  to  directions  given 
under  Total  Sulphates,  p.  384.  Calculate  the  quantity  of  sulphur, 
expressed  as  S03  or  S,  present  in  the  twenty-four  hour  urine 
specimen. 

5.  Total  Sulphur. — Sodium  Hydroxide  and  Potassium  Nitrate 
Fusion  Method. — Place  25  c.c.  of  urine  in  a  silver  crucible  and 
evaporate  to  a  thick  syrup  on  a  water-bath.  Add  10  gram-  of 
sodium  hydroxide  and  2  grams  of  potassium  nitrate  to  the  residue 
and  fuse  the  mass,  over  an  alcohol  flame,  until  all  organic  matter  has 
disappeared  and  the  fused  mixture  is  clear.  Cool  the  mixture. 
transfer  it  to  a  casserole,  by  means  of  hot  water,  acidify  slightly 
with  hydrochloric  acid  and  evaporate  it  to  dryness  on  a  water  bath. 
Moisten  the  residue  with  a  few  drops  of  dilute  hydrochloric  acid 
and  bring  it  into  solution  with  hot  water.  Filter,  heat  the  filtrate 
to  boiling  and  immediately  precipitate  it  by  the  addition  "i   10  c.c. 

xThis  moistening  of  the  residue  with  a  small  amount  of  water  1-  verj  essential 
and    should    not   be   neglected. 
"About  18  c.c.  of  acid  is  required  for  S  grams  of  sodium  peroxide. 
3  See  note  (2)  at  the  bottom  of  page  384. 


388 


PHYSIOLOGICAL    CHEMISTRY. 


of  a  10  per  cent  solution  of  barium  chloride,  adding  the  solution 
slowly,  drop  by  drop.  Allow  the  precipitated  solution  to  stand  2 
hours  and  filter  while  cold.  Ignite,  weigh  and  calculate  according 
to  directions  given  under  Total  Sulphates,  p.  384. 

Compute  the  quantity  of  sulphur,  expressed  as  SOs  or  S,  present 
in  the  twenty-four  hour  urine  specimen. 


Ftg.   124. 


Berthelot-Atwater  Bomb  Calorimeter.      (Cross-section   of  Apparatus  as   Ready 

for  Use.) 

A,  Steel  cup  or  bomb  proper ;  C,  collar  of  steel ;  G,  opening  through  which  oxygen 
is  forced  into  the  bomb  ;  H  and  I',  insulated  wires  which  serve  to  conduct  an  electric 
current  for  igniting  the  substance  which  is  held  in  the  small  capsule ;  L,  a  stirrer 
which  serves  to  keep  the  water  surrounding  the  bomb  in  motion  and  insures  the 
equalization  of  temperature ;  P,  a  delicate  thermometer  which  shows  the  rise  in 
temperature  of  the  water  surrounding  the  bomb. 


URINE:    QUANl  N  \Tl\  I.    ANALYSIS. 

6.  Total  Sulphur. — Sherman's  Compressed  Oxygen  Method.1  — 
Evaporate  as  much  urine  on  an  absorbenl  filter  block2  at  55  1 
the  block  will  conveniently  absorb  and  burn  the  block  so  prepared 
in  a  bomb-calorimeter3  using  25  30  atmospheres  of  oxygen. 
Connect  the  bomb  with  a  wash-bottle  containing  water,  and  allow 
the  gas  to  bubble  through  the  liquid  until  the  high  pressure  within 
the  apparatus  has  been  reduced  to  atmospheric  pressure.  Now  open 
the  bomb  and  thoroughly  rinse  the  interior,  using  water  from  the 
wash-bottle  for  the  first  rinsing.  Dissolve  any  ash  found  in  the 
combustion  capsule  in  hydrochloric  acid  and  add  this  solution  to  the 
main  solution.  Evaporate  to  150  c.c,  filter  and  cool  the  filtrate. 
Add  10  c.c.  of  a  5  per  cent  solution  of  barium  chloride  to  the  cold 
filtrate,  slowly,  drop  by  drop.4  The  contents  of  the  flask  should  not 
be  stirred  or  shaken  during-  the  addition  of  the  barium  chloride. 
Allow  the  mixture  to  stand  at  least  one  hour,  then  shake  up  the 
solution  and  filter  it  through  a  weighed  Gooch  crucible.  Manipu- 
late the  precipitate  of  BaS04  according  to  direction  given  under 
Total  Sulphates,  page  384. 

Calculate  the  quantity  of  sulphur,  expressed  as  SO:J  or  S,  present 
in  the  twenty-four  hour  urine  specimen. 

IX.     Phosphorus. 

1.  Total  Phosphates. — Uranium  Acetate  Method. — To  50  c.c. 
of  urine  in  a  small  beaker  or  Erlenmeyer  flask  add  5  c.c.  of  a  special 
sodium  acetate  solution5  and  heat  the  mixture  to  the  boiling-point. 
From  a  burette,  run  into  the  hot  mixture,  drop  by  drop,  a  standard 
solution  of  uranium  acetate0  until  a  precipitate  ceases  to  form  and 

1  See  Sherman's  Organic  Analysis,  p.  19. 

2  Only  a  small  amount  of  urine  should  be  added  at  one  time,  it  being  nee 

to  make  several  evaporations  before  the  block  contains  sufficient  urinary  residue 
to  proceed  with  the  combustion. 

3 The  Berthelot-Atwater  apparatus  (Fig.  124,  page  388)  is  well  adapted  to  this 
purpose. 

4  See  note  (2)  at  the  bottom  of  page  3X4. 

5  The  sodium  acetate  solution  is  prepared  by  dissolving  100  grams  of  sodium 
acetate  in  800  c.c.  of  distilled  water,  adding  100  c.c.  of  30  per  cent  acetic  acid  to 
the  solution  and  making  the  volume  of  the  mixture  up  to   1   liter  with  water. 

"This  uranium  acetate  solution  may  he  prepared  by  dissolving  35401  grams 
of  uranium  acetate  in  one  liter  of  water.  One  c.c.  of  such  a  solution  should  be 
equivalent  to  0.005  gram  of  P-Oe,  phosphoric  anhydride.  This  solution  may  be 
standardized  as  follows:  To  51'  c.c.  of  a  standard  solution  of  disodium  hyd 
phosphate,  of  such  a  strength  that  the  50  c.c.  contains  0.1  gram  of  PjO§,  add 
5  c.c.  of  the  sodium  acetate  solution,  mentioned  above,  and  titrate  with  the 
uranium  solution  to  the  correct  end-reaction  as  indicated  in  the  method  proper. 


390  PHYSIOLOGICAL    CHEMISTRY. 

a  drop  of  the  mixture  when  removed  by  means  of  a  glass  rod  and 
brought  in  contact  with  a  drop  of  a  solution  of  potassium  ferrocya- 
nide  on  a  porcelain  test-tablet  produces  instantaneously  a  brownish- 
red  coloration.1  Take  the  burette  reading  and  calculate  the  P205 
content  of  the  urine  under  examination. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uran- 
ium acetate  solution  used  by  0.005  to  determine  the  number  of 
grams  of  P205  in  the  50  c.c.  of  urine  used.  To  express  the  result 
in  percentage  of  P205  multiply  the  value  just  obtained  by  2,  e.  g., 
if  50  c.c.  of  urine  contained  0.074  gram  of  P205  it  would  be  equiva- 
lent to  0.148  per  cent. 

Calculate,  in  terms  of  P205,  the  total  phosphate  content  of  the 
twenty-four  hour  urine  specimen. 

2.  Earthy  Phosphates. — To  100  c.c.  of  urine  in  a  beaker  add  an 
excess  of  ammonium  hydroxide  and  allow  the  mixture  to  stand 
12-24  hours.  Under  these  conditions  the  phosphoric  acid  in  com- 
bination with  the  alkaline  earths,  calcium  and  magnesium,  is  pre- 
cipitated as  phosphates  of  these  metals.  Collect  the  precipitate 
on  a  filter  paper  and  wash  it  with  very  dilute  ammonium  hydroxide. 
Pierce  the  paper,  and  remove  the  precipitate  by  means  of  hot  water. 
Bring  the  phosphates  into  solution  by  adding  a  small  amount  of 
dilute  acetic  acid  to  the  warm  solution.  Make  the  volume  up  to 
50  c.c.  with  water,  add  5  c.c.  of  sodium  acetate  solution  and  de- 
termine the  P2Os  content  of  the  mixture  according  to  the  directions 
given  under  the  previous  method. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uran- 
ium acetate  solution  used  by  0.005  to  determine  the  number  of 
grams  of  P205  in  the  100  c.c.  of  urine  used.  Since  100  c.c.  of 
urine  was  taken  this  value  also  expresses  the  percentage  of  P205 
present. 

Calculate  the  quantity  of  earthy  phosphates,  in  terms  of  P2Os, 
present  in  the  twenty-four  hour  urine  specimen. 

The  quantity  of  phosphoric  acid  present  in  combination  with  the 
alkali  metals  may  be  determined  by  subtracting  the  content  of 
earthy  phosphates   from  the  total  phosphates. 

3.  Total  Phosphorus. — Sodium  Hydroxide  and  Potassium  Nit- 

Inasmuch  as  1  c.c.  of  the  uranium  solution  should  precipitate  0.005  gram  of 
P2O5,  exactly  20  c.c.  of  the  uranium  solution  should  be  required  to  precipitate 
50  c.c.  of  the  standard  phosphate  solution.  If  the  two  solutions  do  not  bear 
this  relation  to  each  other  they  may  be  brought  into  proper  relation  by  diluting 
the  uranium  solution  with  distilled  water  or  by  increasing  its  strength. 
XA  ten  per  cent  solution  of  potassium  ferrocyanide  is  satisfactory. 


urine:  quantitative  analysis. 

rate  Fusion  Method. — Place  25  i-.r.  of  urine  in  a  large  silver  cru- 
cible and  evaporate  to  a  syrup  on  a  water-bath.  \<1<1  i<>  gram- 
of  NaOIl  and  2  grains  of  KXO:,  to  the  residue  and  fuse  the  mass 
until  all  organic  matter  has  disappeared  and  the  fused  mixture  is 
clear.  Cool  the  mixture,  transfer  it  to  a  casserole  by  means  of  hoi 
water,  acidify  the  solution  slightly  with  purr  nitric  acid  and  evapo- 
rate to  dryness  on  a  water-bath.  Moisten  the  residue  with  a 
drops  of  dilute  nitric  acid,  dissolve  it  in  hoi  water  and  transfer  to 
a  beaker.  Now  add  an  equal  volume  <>\  molyhdic  solution1  and 
keep  the  mixture  at  40  C.  for  twenty-four  hours.  Filter  off  the 
precipitate,  wash  it  with  dilute  molyhdic  solution  and  dissolve  it  in 
dilute  ammonia.  Add  dilute  hydrochloric  acid  to  the  solution,  being 
careful  to  leave  the  solution  distinctly  ammoniacal.  Magnesia  mix- 
ture2 (10-15  c-c-)  should  now  be  added  and  after  stirring  thorough- 
ly and  making  strongly  ammoniacal  with  concentrated  ammonia 
the  solution  should  be  allowed  to  stand  in  a  cool  place  for  twenty- 
four  hours.  Filter  off  the  precipitate,  wash  it  free  from  chlorine  by 
means  of  dilute  ammonia  (1:5),  dry,  incinerate  and  weigh,  as 
magnesium  pyrophosphate,  Mg2P207,  in  the  usual  manner. 

In  this  method  the  phosphoric  acid  of  the  urine  is  precipitated  as 
ammonium  magnesium  phosphate  and  in  the  process  of  incinera- 
tion this  body  is  transformed  into  magnesium  pyrophosphate. 

Calculation, — The  quantity  of  phosphorus,  expressed  in  terms 
of  P2Os,  in  the  volume  of  urine  taken  may  be  determined  by  means 
of  the  following  proportion : 

Mol.  wt.  Wt.  of  Mol.  wt. 

Mg2P207 :  Mg2P207 :  :P2C>5  :  x  (wt.  of  P2Ofi  in  grams), 
ppt. 

If  y  represents  the  weight  of  the  Mg2P207  precipitate  and  we 
make  the  proper  substitutions  we  have  the  following  proportion: 

221. 1  :  y : :  140.9:  x   (wt.   of   P205,   in  grams,   in   the   quantity   of 

urine  used.) 

To  express  the  result  in  percentage  of  P20B  simply  divide  the 
value  of  x,  as  just  determined,  by  the  quantity  of  urine  used. 

1  Directions  for  the  preparation  of  the  solution  arc  given  on  p.  57- 

2  Directions  for  the  preparation  of  magnesia  mixture  may  be  found  on  p 


392  PHYSIOLOGICAL    CHEMISTRY. 

i 

X.     Creatinine. 

Folin's  Colorimetric  Method. — This  method  is  based  upon  the 
characteristic  property  possessed  alone  by  creatinine,  of  yielding  a 
certain  definite  color-reaction  in  the  presence  of  picric  acid  in 
alkaline  solution.  The  procedure  is  as  follows:  Place  10  c.c.  of 
urine  in  a  500  c.c.  volumetric  flask,  add  15  c.c.  of  a  saturated  solu- 
tion of  picric  acid  and  5  c.c.  of  a  10  per  cent  solution  of  sodium 
hydroxide,  shake  thoroughly  and  allow  the  mixture  to  stand  for 
5  minutes.  During  this  interval  pour  a  little  §  potassium  bichro- 
mate solution1  into  each  of  the  two  cylinders  of  the  color- 
imeter (Duboscq's)  and  carefully  adjust  the  depth  of  the 
solution  in  one  of  the  cylinders  to  the  8  mm.  mark.  A  few  pre- 
liminary colorimetric  readings  may  now  be  made  with  the  solution 
in  the  other  cylinder,  in  order  to  insure  greater  accuracy  in  the  sub- 
sequent examination  of  the  solution  of  unknown  strength.  Obvi- 
ously the  two  solutions  of  potassium  bichromate  are  identical  in 
color  and  in  their  examination  no  two  readings  should  differ  more 
than  0.1-0.2  mm.  from  the  true  value  (8  mm.).  Four  or  more 
readings  should  be  made  in  each  case  and  an  average  taken  of  all 
of  them  exclusive  of  the  first  reading,  which  is  apt  to  be  less  ac- 
curate than  the  succeeding  readings.  In  time  as  one  becomes  pro- 
ficient in  the  technique  it  is  perfectly  safe  to  take  the  average  of 
the  first  huo  readings. 

At  the  end  of  the  5-6  minute  interval  already  mentioned,  the  con- 
tents of  the  500  c.c.  flask  are  diluted  to  the  500  c.c.  mark,  the  bichro- 
mate solution  is  thoroughly  rinsed  out  of  one  of  the  cylinders  and 
replaced  with  the  solution  thus  prepared  and  a  number  of  colorimet- 
ric readings  are  immediately  made. 

Ordinarily  10  c.c.  of  urine  is  used  in  the  determination  by  this 
method  but  if  the  content  of  creatinine  is  above  15  mg.  or  below 
5  mg.  the  determination  should  be  repeated  with  a  volume  of 
urine  selected  according  to  the  content  of  creatinine.  This  variation 
in  the  volume  of  urine  according  to  the  content  of  creatinine  is  quite 
essential,  since  the  method  loses  in  accuracy  when  more  than  15 
mg.  or  less  than  5  mg.  of  creatinine  is  present  in  the  solution  of 
unknown  strength. 

Calculation. — By  experiment  it  has  been  determined  that  10  mg. 
of  pure  creatinine,  when  brought  into  solution  and  diluted  to  500 
c.c.  as  explained  in  the  above  method,  yields  a  mixture  8.1  mm. 
of  which  possesses  the  same  colorimetric  value  as  8  mm.   of  a 

1  This  solution  contains  24.55  grams  of  potassium  bichromate  to  the  liter. 


urine:  quantitative  analysis.  393 

f  solution  of  potassium  bichromate*.  ('.caring  this  in  mind  the  com 
putation  is  readily  ma.K-  l»y  means  of  the  following  proportion  in 
which  y  represents  the  number  of  mm.  of  the  solution  of  unknown 
strength  equivalent  to  the  8  nun.  of  the  potassium  bichromate  solu- 
tion : 

y.8.1::  io:.v  (mgs.  of  creatinine  in  the  quantity  of  urine  used). 

This  proportion  may  be  used  for  the  calculation  no  matter  what 
volume  of  urine  (5,  10  or  15  c.c. )  is  used  in  the  determination. 
The  10  represents  10  mg.  of  creatinine  which  gives  a  color  equal 
to  8.1  mm.,  whether  dissolved  in  5,  to  or  [5  c.c.  of  fluid. 

Calculate  the  quantity  of  creatinine  in  the  twenty-four  hour  urine 
specimen. 

XI.     Creatine. 

Folin-Benedict  Method. — To  about  20  c.c.  of  urine  in  a  50  c.c. 
volumetric  flask,  add  20  c.c.  of  normal  hydrochloric  acid  and  place 
the  flask  in  an  autoclave  at  a  temperature  of  117-1200  C.  for  one- 
half  hour.  Add  distilled  water  until  the  volume  of  the  acid- 
urine  mixture  is  exactly  50  c.c,  close  the  flask  by  means  of  a  stopper, 
and  shake  it  thoroughly.  Approximately  neutralize  25  c.c.  of  this 
mixture,  introduce  it  into  a  500  c.c.  volumetric  flask  and  deter- 
mine its  creatinine  content  according  to  Folin's  Method  (see  p.  31  12  1 . 

Calculation. — Calculate  as  explained  on  p.  392,  and  from  this 
value  substract  the  value  for  the  original  content  of  creatinine  before 
hydrolysis.  The  difference  between  these  two  values  will  be  the 
creatine  content  of  the  original  urine  in  terms  of  creatinine. 

XII.     Indican. 

Ellinger's  Method.— This  method  for  the  quantitative  determin- 
ation of  indican  is  based  upon  the  principle  underlying  Jaffe's  test 
for  the  detection  of  indican  (see  p.  280).  The  method  is  as  f<  'Hi  iws  : 
To  50  c.c.  of  urine1  in  a  small  beaker  or  casserole  add  5  c.c.  of 
basic  lead  acetate  solution,  mix  thoroughly  and  Alter.  Transfer  40 
c.c.  of  the  filtrate  to  a  separatory  funnel,  add  an  equal  volume  of 
Obermaver's  reagent  (see  p.  281)  and  20  c.c.  of  chloroform  and 
extract  in  the  usual  manner.  This  extraction  with  chloroform 
should  be  repeated  until  the  chloroform  solution  remains  colorless. 
Now  filter  the  combined  chloroform  extracts  through  a  dry  filter 

lIf  the  urine  under  examination  is  neutral  or  alkaline  in  reaction  it  should  he 
made   faintly  acid   with    acetic   acid   before   adding   the   basic   lead    acetate. 


394  PHYSIOLOGICAL    CHEMISTRY. 

paper  into  a  dry  Erlenmeyer  flask.  Distil  off  the  chloroform,  heat 
the  residue  on  a  boiling  water-bath  for  5  minutes  in  the  open  flask, 
and  wash  the  dried  residue  with  hot  water.1  Add  10  c.c.  of  con- 
centrated sulphuric  acid  to  the  washed  residue,  heat  on  the  water- 
bath  for  5-10  minutes,  dilute  with  100  c.c.  of  water  and  titrate  the 
blue  solution  with  a  very  dilute  solution  of  potassium  permangan- 
ate.2 The  end-point  is  indicated  by  the  dissipation  of  all  the  blue 
color  from  the  solution  and  the  formation  of  a  pale  yellow  color. 

Calculation. — Ellinger  claims  that  one-sixth  of  the  amount  deter- 
mined must  be  added  to  the  value  obtained  by  titration  in  order 
to  secure  accurate  data.     This  correction  should  always  be  made. 

XIII.     Chlorides. 

1.  Clark's  Modification  of  Dehn's  Method.3 — In  this  method 
the  organic  compounds,  that  hold  the  chlorine  too  firmly  for  its 
quantitative  precipitation  with  argentic  nitrate,  are  destroyed  by 
oxidation  with  sodium  peroxide.  Sodium  peroxide  in  the  pres- 
ence of  water  gives  off  nascent  oxygen  according  to  the  following 
equation. 

Na202  +  H20  =2NaOH  +  O 

The  oxygen  then  attacks  the  organic  matter  and  the  chlorine  is 
left  as  sodium  chloride.  The  procedure  is  as  follows :  To  10  c.c. 
of  urine  in  a  75-100  c.c.  casserole,  add  1. 0-1.2  gram  of  sodium 
peroxide  and  evaporate  the  mixture  to  dryness  on  a  boiling  water- 
bath.  In  case  the  residue  is  not  pure  white,  thus  indicating  that  in- 
sufficient sodium  peroxide  has  been  added,  the  residue  should  be 
moistened  with  distilled  water,  additional  sodium  peroxide  added, 
and  the  mixture  again  evaporated  to  dryness.  When  the  oxidation 
is  complete,  treat  the  mass  with  10-20  c.c.  of  distilled  water  and  stir 
until  it  has  practically  all  been  brought  into  solution.  Then  intro- 
duce a  bit  of  litmus  paper  and  add  dilute  nitric  acid  (1:1)  until  the 
litmus  paper  turns  red  and  all  effervescence  ceases.     Now  place  the 

1  The  washing  should  be  continued  until  the  wash  water  is  no  longer  colored. 
Ordinarily  two  or  three  washings  are  sufficient.  If  a  separation  of  indigo 
particles  takes  place  during  the  washing  process,  the  wash  water  should  be 
filtered,  the  indigo  extracted  with  chloroform  and  the  usual  method  applied 
from  this  point. 

2  A  "  stock  solution "  of  potassium  permanganate  containing  three  grams  per 
liter  should  be  prepared,  and  when  needed  for  titration  purposes  a  suitable 
volume  of  this  solution  should  be  diluted  with  40  volumes  of  water.  The 
potassium  permanganate  solution  should  be  standardized  with  pure  indigo. 

3  Private  communication  to  the  author  from  Mr.  S.  C.  Clark. 


urine:   QUANTITATIVE    ANALYS] 

casserole  on  a  hot  plate  or  on  a  gauze  and  heal  the  contents  almost 
to  the  boiling-point.3     To  the  hoi  solution  add  a  standard  solution  of 

argentic  nitrate  (see  page  396)  in  slight  excess.2      Filter  off  fi- 
ver chloride  while  the  solution  is  still  hot  and  wash  the  precipitate 

thoroughly  with  distilled  water.  To  the  lilt  rate,  add  1  <■..-.  of  a 
saturated  solution  of  ferric  ammonium  sulphate  and  then  titrate  with 
a  standard  solution  of  ammonium  thiocyanate  1  see  page  ,v;7)  until 
the  clear,  slightly  yellow  fluid  (or  the  opalescent,  milky  thud,  in 
case  there  is  much  excess  of  argentic  nitrate)  change-  to  a  slight  red 
dish-brown  color.  The  color  of  the  end-point  varies  with  the  in- 
dividual. The  exact  end-point  reached  is  not  so  important  a-  1-  t he- 
securing  of  the  same  end-point  in  a  series  of  determinations  as 
that  obtained  in  the  standardization    of  the  standard  solutions  used. 

Calculation. — The  standard  solution  of  argentic  nitrate  should  be 
made  up  so  that  I  c.c.  equals  0.010  gram  of  sodium  chloride  and 
1  c.c.  of  the  ammonium  thiocyanate  should  he  equivalent  to  1  c.c.  of 
the  argentic  nitrate  solution  (see  pp.  396  and  397).  Then,  if  the 
number  of  cubic  centimeters  of  ammonium  thiocyanate  u-c<\  he  sub- 
tracted from  the  number  of  cubic  centimeters  of  argentic  nitrate, 
the  difference  is  the  number  of  cubic  centimeters  of  argentic  nitrate 
actually  used  in  the  precipitation  of  chlorine  as  silver  chloride. 
This  number,  multiplied  by  0.010,  gives  the  weight  in  grams  of  the 
sodium  chloride  in  the  10  c.c.  of  urine  used.  If  it  is  desired  to 
express  the  result  in  percentage  of  sodium  chloride,  move  the  deci- 
mal point  one  place  to  the  right. 

In  a  similar  manner  the  weight  or  percentage  of  chlorine  may  be 
computed,  using  the  factor  0.006  as  explained  in  Mohr's  method, 
page  396.  Calculate  the  quantity  of  sodium  chloride  and  of  chlo- 
rine in  the  twenty-four  hour  urine  specimen. 

2.  Mohr's  Method. — To  10  c.c.  of  urine  in  a  small  platinum  or 
porcelain  crucible  or  dish  add  about  2  grams  of  chlorine-free  potas- 
sium nitrate  and  evaporate  to  dryness  at  100 °  C.  (The  evapora- 
tion may  be  conducted  over  a  low  flame  provided  care  is  taken  to 
prevent  loss  by  spurting.)  By  means  of  crucible  tongs  hold  the 
crucible  or  dish  over  a  free  flame  until  all  carbonaceous  matter  has 
disappeared  and  the  fused  mass  is  slightly  yellow  in  color.  Cool  the 
residue  somewhat  and  bring  it  into  solution  in  a  small  amount   1  1  g 

xIf  there  is  a  slight  precipitate,  due  to  silicic  acid  from  the  casserole,  this  is 
filtered  ofif  and  the  filtrate  collected  in  a  200  c.c.  beaker. 

2  This  point  is  most  easily  recognized  by  keeping  the  solution  h"t  and  in 
constant  agitation  while  adding  the  argentic  nitrate  so  that  the  silver  chloride 
formed  coagulates  and  sinks,  leaving  a  clear,  supernatant   fluid. 


396  PHYSIOLOGICAL    CHEMISTRY. 

25  c.c.)  of  distilled  water  acidified  with  about  10  drops  of  nitric 
acid.  Transfer  the  solution  to  a  small  beaker,  being  sure  to  rinse 
out  the  crucible  or  dish  very  carefully.  Test  the  reaction  of  the 
fluid,  and  if  not  already  acid  in  reaction  to  litmus,  render  it  slightly 
acid  with  nitric  acid.  Now  neutralize  the  solution  by  the  addition 
of  calcium  carbonate  in  substance,1  add  2-5  drops  of  neutral  potas- 
sium chromate  solution  to  the  mixture  and  titrate  with  a  standard 
argentic  nitrate  solution.2 

This  standard  solution  should  be  run  in  from  a  burette,  stirring 
the  liquid  in  the  beaker  after  each  addition.  The  end-reaction  is 
reached  when  the  yellow  color  of  the  solution  changes  to  a  slight 
orange-red.  At  this  point  take  the  burette  reading  and  compute 
the  percentage  of  chlorine  and  sodium  chloride  in  the  urine  ex- 
amined. 

Calculation. — Since  1  c.c.  of  the  standard  argentic  nitrate  solu- 
tion is  equivalent  to  0.010  gram  of  sodium  chloride,  to  obtain  the 
weight,  in  grams,  of  the  sodium  chloride  in  the  10  c.c.  of  urine 
used  multiply  the  number  of  cubic  centimeters  of  standard  solu- 
tion used  by  0.0 10.  If  it  is  desired  to  express  the  result  in  per- 
centage of  sodium  chloride  move  the  decimal  point  one  place  to 
the  right. 

To  obtain  the  zveight,  in  grams,  of  the  chlorine  in  the  10  c.c.  of 
urine  used  multiply  the  number  of  cubic  centimeters  of  standard 
solution  used  by  0.006,  and  if  it  is  desired  to  express  the  result 
in  percentage  of  chlorine  move  the  decimal  point  one  place  to  the 
right. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the 
twenty- four  hour  urine  specimen. 

3.  Volhard-Arnold  Method. — Place  10  c.c.  of  urine  in  a  100 
c.c.  volumetric  flask,  add  20-30  drops  of  nitric  acid  (sp.  gr.  1.2) 
and  2  c.c.  of  a  cold  saturated  solution  of  ferric  alum.  If  necessary, 
at  this  point  a  few  drops  of  an  8  per  cent  solution  of  potassium 
permanganate  may  be  added  to  dissipate  the  red  color.  Now  slow- 
ly run  in  the  standard  argentic  nitrate3  solution  (20  c.c.  is  ordi- 
narily used)  until  all  the  chlorine  has  been  precipitated  and  an  excess 

1  The  cessation  of  effervescence  and  the  presence  of  some  undecomposed  cal- 
cium carbonate  at  the  bottom  of  the  vessel  are  the  indications  of  neutralization. 

2  Standard  argentic  nitrate  solution  may  be  prepared  by  dissolving  29.060  grams 
of  argentic  nitrate  in  1  liter  of  distilled  water.  Each  cubic  centimeter  of  this 
solution  is  equivalent  to  0.010  gram  of  sodium  chloride  or  to  0.006  gram  of 
chlorine. 

3  See  note  (2)  at  the  bottom  of  page  384. 


urine:  quantitative  ANALYS] 

of  the  argentic  nitrate  solution  is  present,  continually  shaking  the 
mixture  during  the  addition  of  the  standard  solution.  Allow  the 
flask  to  -.land  10  minutes,  thru  fill  n  to  the  coo  c.c.  graduation 
with  distilled  water  and  thoroughly  mix  the  contents.  Now  filter 
the  mixture  through  a  dry  filter  paper,  colled  50  c.c.  of  the  filtrate 
and  titrate  it  with  standardized  ammonium  thiocyanate  solution.1 
The  first  permanent  tinge  of  brown  indicates  the  end-point.  Take 
the  burette  reading  and  compute  the  weight  of  sodium  chloride  in 
the  10  c.c.  of  urine  used. 

Calculation. — The  number  of  cubic  centimeters  of  ammonium 
thiocyanate  solution  used  indicates  the  excess  of  standard  argentic 
nitrate  solution  in  the  50  c.c.  of  filtrate  titrated.  Multiply  this 
reading  by  2,  inasmuch  as  only  one-half  of  the  filtrate  was  employed, 
and  subtract  this  product  from  the  number  of  cubic  centimeter-  of 
argentic  nitrate  (20  c.c.)  originally  used,  in  order  to  obtain  the 
actual  number  of  cubic  centimeters  of  argentic  nitrate  utilized  in 
the  precipitation  of  the  chlorides  in  the  10  c.c.  of  urine  employed. 

To  obtain  the  weight  in  grams,  of  the  sodium  chloride  in  the 
10  c.c.  of  urine  used  multiply  the  number  of  cubic  centimeter-  of 
the  standard  argentic  nitrate  solution,  actually  utilized  in  the  pre- 
cipitation, by  0.010.  If  it  is  desired  to  express  the  result  in  per- 
centage of  sodium  chloride  move  the  decimal  point  one  place  to  the 
right. 

In  a  similar  manner  the  weight,  or  percentage  of  chlorine  may 
be  computed  using  the  factor  0.006  as  explained  in  Mohr's  method. 
page  396. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the 
twenty-four  hour  urine  specimen. 

XIV.     Acetone  and  Diacetic  Acid. 
1.  Folin-Hart  Method. — This  method  serves  the  same  purpose 
as  the  Messinger-Huppert  Method,  /*.  c,  the  determination  of  both 

'This  solution  is  made  of  such  a  strength  that  I  c.c.  of  it  is  equal  to  i  c.c.  of 
the  standard  argentic  nitrate  solution  used.  To  prepare  the  solution  di« 
12.9  grams  of  ammonium  thiocyanate,  NHiSCN,  in  a  little  less  than  a  liter 
of  water.  In  a  small  flask  place  20  c.c.  of  the  standard  argentic  nitrate  solution. 
5  c.c.  of  the  ferric  alum  solution  and  4  c.c.  of  nitric  acid  (sp.  gr.  r.2),  add  water 
to  make  the  total  volume  100  c.c.  and  thoroughly  mix  the  contents  of  the  flask. 
Now  run  in  the  ammonium  thiocyanate  solution  from  a  burette  until  a 
permanent  brown  tinge  is  produced.  This  is  the  end-reaction  and  indicates  that 
the  last  trace  of  argentic  nitrate  has  been  precipitated.  Take  the  burette  reading 
and  calculate  the  amount  of  water  necessary  to  use  in  diluting  the  ammonium 
thiocyanate  in  order  that  10  c.c.  of  this  solution  may  be  exactly  equal  to 
10  c.c.  of  the  argentic  nitrate  solution.  Make  this  dilution  and  titrate  again  to 
be  certain  that  the  solution  is  of  the  proper  strength. 


39<3  PHYSIOLOGICAL    CHEMISTRY. 

acetone  and  diacetic  acid  in  terms  of  acetone.  It  is,  however,  much 
simpler  and  less  time  consuming.  The  method  includes  the 
transformation  of  the  diacetic  acid  into  acetone  and  carbon  dioxide 
by  means  of  heat  and  the  subsequent  removal  of  the  acetone  thus 
formed  as  well  as  the  preformed  acetone  by  means  of  an  air  current 
as  first  suggested  by  Folin  (see  p.  400).  The  procedure  is  as  fol- 
lows :  Introduce  into  a  wide-mouthed  bottle  200  c.c.  of  water,  an 
accurately  measured  excess  of  j-q  iodine  solution1  and  an  excess  of 
40  per  cent  potassium  hydroxide.  Prepare  an  aerometer  cylinder 
containing  alkaline  hypoiodite  solution  to  absorb  any  acetone  which 
may  be  present  in  the  air  of  the  laboratory  and  between  the  cylinder 
and  bottle  suspend  a  test-tube  about  two  inches  in  diameter.  This 
large  test-tube  should  contain  20  c.c.  of  the  urine  under  examina- 
tion, 10  drops  of  a  ten  per  cent  solution  of  phosphoric  acid,  10 
grams  of  sodium  chloride,  and  a  little  petroleum,  and  should  be 
raised  sufficiently  high  to  facilitate  the  easy  application  of  heat  to 
its  bottom  portion.  The  connections  on  the  side  of  the  tube  should 
be  provided  with  bulb  tubes  containing  cotton.  When  the  appa- 
ratus is  arranged  as  described,  it  should  be  connected  with  a  Chap- 
man pump  and  an  air  current  passed  through  for  twenty-five  min- 
utes. During  this  period  the  contents  of  the  test-tube  are  heated 
just  to  the  boiling-point  and  after  an  interval  of  five  minutes  again 
heated  in  the  same  manner.  By  this  means  the  diacetic  acid  is  con- 
verted into  acetone  and  at  the  end  of  the  twenty-five  minute  period 
this  acetone,  as  well  as  the  preformed  acetone,  will  have  been  re- 

1  Proceed  as  follows  in  order  to  obtain  a  rough  idea  regarding  the  amount  of 
~  iodine  solution  to  be  used :  Introduce  into  a  test-tube  10  c.c.  of  the  urine 
under  examination  and  1  c.c.  of  a  solution  of  ferric  chloride  made  by  dissolving 
100  grams  of  ferric  chloride  in  100  c.c.  of  distilled  water.  After  permitting  the 
mixture  to  stand  for  two  minutes,  compare  the  color  with  that  of  an  equal 
volume  of  the  ferric  chloride  solution  in  a  test-tube  of  similar  diameter.  If 
the  two  solutions  be  of  approximately  the  same  color  intensity,  20  c.c.  of  the 
urine  under  examination  will  yield  sufficient  acetone  to  require  nearly  10  c.c. 
of  -^j  iodine  solution.  In  case  the  mixture  is  darker  in  color  than  is  the 
ferric  chloride  solution,  the  former  should  be  diluted  with  distilled  water  until 
it  is  of  approximately  the  same  intensity  as  the  ferric  chloride  solution.  From 
this  data  the  amount  of  -^  iodine  solution  required  may  be  roughly  estimated 
by  means  of  the  following  table : 


Urine  c.c. 

Ferric  Chloride. 

Water. 

^   Iodine  Required  c.c. 

IO 
IO 
IO 
IO 

I 
I 
I 
I 

IO 
20 
30 

IO 
20 

35 
5o 

urine:  quantitative  analysis. 

moved  from  the  urine  to  the  absorption  bottle  and  there  retained 

as  iodoform. 

The  contents  of  the  absorption  bottle  should  now  be  acidified  with 
concentrated  hydrochloric  acid,]  and  titrated  with  ,N,,  sodium  thio- 
sulphate  and   starch   as   in    the    Messinger  Hupperl    method 

below). 

2.  Messinger-Huppert  Method.2 — Place  [00  c.c.  of  urine  in  a 

distillation  flask  and  add  j  c.c.  of  50  per  cenl  acetic  acid.     '  !onned 
the  flask  with  a  condenser,  properly  arrange  a   receiver,  attach  a 
terminal  series  of  bulbs  containing  water  and  distil  over  aboul  nine 
tenths  of  the  urine  mixture.     Remove  the  receiver,  attach  another 

and  subject  the  residual  portion  of  the  mixture  to  a  second  distil- 
lation. Test  this  fluid  for  acetone  and  if  the  presence  of  acetone 
is  indicated  add  about  ioo  c.c.  of  water  to  the  residue  and  again 
distil.  Treat  the  united  acetone  distillates  with  i  c.c.  of  dilute  I  i  _' 
per  cent)  sulphuric  acid  and  redistil,  collecting  this  second  distillate 
in  a  glass-stoppered  flask.  During  distillation,  however,  the  glass 
stopper  is  replaced  by  a  cork  with  a  double  perforation,  the  glass 
tube  from  one  perforation  passing  to  the  condenser,  while  the  bulbs 
containing  water,  before  mentioned,  are  attached  by  means  of  the 
tube  in  the  other  perforation.  Allow  the  distillation  process  to  pro- 
ceed until  practically  all  of  the  fluid  has  passed  over,  then  remove 
the  receiving  flask  and  insert  the  glass  stopper.  Now  treat  the  dis- 
tillate carefully  with  10  c.c.  of  a  y$  solution  of  iodine  and  add 
sodium  hydroxide  solution,  drop  by  drop,  until  the  blue  color  is  dis- 
sipated and  the  iodoform  precipitates.  Stopper  the  flask  and  shake 
it  for  about  one  minute,  acidify  the  solution  with  concentrated 
hydrochloric  acid,  and  note  the  production  of  a  brown  color  if  an 
excess  of  iodine  is  present.  In  case  there  is  no  such  excess,  the 
solution  should  be  treated  with  tNq  iodine  solution  until  an  excess 
is  obtained.  Retitrate  this  excess  of  iodine  with  ,N,,  sodium  thio- 
sulphate  solution  until  a  light  yellow  color  is  observed.  At  this 
point  a  few  cubic  centimeters  of  starch  paste  should  he  added  and 
the  mixture  again  titrated  until  no  blue  color  is  visible.  This  is 
the  end-reaction. 

Calculation.  —  Subtract  the  number  of  cubic  centimeters  of  ,N„ 
thiosulphate  solution  used  from  the  volume  of  ,x,,  iodine  solution 
employed.      Since    i    c.c.   of   the   iodine   solution    is   equivalent   to 

1  An  excess  of  iodine  is  indicated  by  the  development  of  a  brown  color 
'This  method  serves  to  determine  both  acetone  and  diacetic  acid  in   terms  of 
acetone. 


400  -PHYSIOLOGICAL    CHEMISTRY. 

0.967  milligram  of  acetone,  and  since  1  c.c.  of  the  thiosulphate 
solution  is  equivalent  to  1  c.c.  of  the  iodine  solution,  if  we  multiply 
the  remainder  from  the  above  subtraction  by  0.967  we  will  obtain 
the  number  of  milligrams  of  acetone  in  the  100  c.c.  of  urine 
examined. 

Calculate  the  quantity  of  acetone  in  the  twenty- four  hour  urine 
specimen. 

XV.     Acetone. 

1.  Folin's  Method. — The  same  type  of  apparatus  is  used  in  this 
method  as  that  described  in  Folin's  method  for  the  determination 
of  ammonia  (see  p.  380).  The  procedure  is  as  follows:  Introduce 
20—25  c.c.  of  the  urine  under  examination  into  the  aerometer  cylinder 
and  add  10  drops  of  10  per  cent  phosphoric  acid,1  8-10  grams  of 
sodium  chloride,2  and  a  little  petroleum.  Introduce  into  an  absorp- 
tion flask,3  such  as  is  used  in  the  ammonia  determination  (see  p. 
380),  150  c.c.  of  water,  10  c.c.  of  a  40  per  cent  solution  of  potassium 
hydroxide,  and  an  excess  of  a  T\  iodine  solution.  Connect  the  flask 
with  the  aerometer  cylinder,  attach  a  Chapman  pump  and  permit  an 
air  current,  slightly  less  rapid  than  that  used  for  the  determination 
of  ammonia,  to  be  drawn  through  the  solution  for  20-25  minutes. 
All  of  the  acetone  will,  at  this  point,  have  been  converted  into  iodo- 
form in  the  absorption  flask.  Add  10  c.c.  of  concentrated  hydro- 
chloric acid  (a  volume  equivalent  to  that  of  the  strong  alkali  orig- 
inally added),  to  the  contents  of  the  latter  and  titrate  the  excess 
of  iodine  by  means  of  ^  sodium  thiosulphate  solution  and  starch, 
as  in  the  Messinger-Huppert  method  (see' p.  399). 

Folin  has  further  made  suggestions  regarding  the  simultaneous 
determination  of  acetone  and  ammonia  by  the  use  of  the  same  air 
current.4  This  is  an  important  consideration  for  the  clinician  inas- 
much as  urines  which  contain  acetone  and  diacetic  acid  are  gener- 
ally those  from  which  the  ammonia  data  are  also  desired.  The  pro- 
cedure for  the  combination  method  is  as  follows :  Arrange  the 
ammonia  apparatus  as  usual  (see  p.  380),  and  to  the  aerometer  of 
the  ammonia  apparatus  attach  the  acetone  apparatus  set  up  as  de- 
scribed above.     Regulate  the  air  current  with  special  reference  to 

1  Oxalic  acid   (0.2-0.3  gram)  may  be  substituted  if  desired. 

2  Acetone  is  insoluble  in  a  saturated  solution  of  sodium  chloride. 

3  Folin's  improved  absorption  tube  (see  Fig.  123,  p.  381)  should  be  used  in  this 
connection  inasmuch  as  the  original  type  embracing  the  use  of  a  rubber  stopper  is 
unsatisfactory  because  of  the  solvent  action  of  alkaline  hypoiodite  on   rubber. 

4  These  determinations  may  even  be  made  on  the  same  sample  of  urine  if  the 
sample  is  too  small  for  the  double  determination. 


urine:  quantitative  analysis.  401 

the  determination  of  acetone  and  at  the  cud  of  -'<)  25  minutes  dis- 
connect the  acetone  apparatus  and  complete  the  determination  of 
the  acetone  as  just  described.  The  air  current  is  not  interrupted 
and  after  having  run  one  and  one  dial  f  hours  the  ammi  >nia  apparatus 
is  detached  and  the  ammonia  determination  completed  as  described 
on  page  380. 

If  data  regarding-  diacetic  acid  are  desired,  the  result  obtained  by 
Folin's  method  may  be  subtracted  from  the  result  obtained  by  the 
Messinger-Huppert  method  (see  p.  399),  inasmuch  as  the  latter 
method  determines  both  acetone  and  diacetic  acid.  Under  all  con- 
ditions the  determination  of  acetone  should  he  as  expedition-  as 
possible.  This  is  essential,  not  only  because  of  the  fact  that  any 
diacetic  acid  present  in  the  urine  will  become  transformed  into 
acetone  but  also  because  of  the  rapid  spontaneous  decomposition  of 
the  alkaline  hypoiodite  solution  used  in  the  determination  of  the 
acetone.  It  has  been  claimed  that  alkaline  hypoiodite  solutions  are 
almost  completely  converted  into  iodate  solutions  in  one-half  hour. 
Folin  states,  however,  that  the  transformation  is  not  so  rapid  as 
this,  but  he  nevertheless  emphasizes  the  necessity  of  rapidity  of 
manipulation.  At  the  same  time  it  should  be  remembered  that  the 
air  current  must  not  be  as  rapid  as  for  ammonia,  inasmuch  as  the 
alkaline  hypoiodite  solution  will  not  absorb  all  the  acetone  under 
those  conditions. 

XVI.     Diacetic  Acid. 

1.  Folin-Hart  Method. — Arrange  the  apparatus  as  described 
under  the  Folin-Hart  method  for  the  determination  of  acetone 
and  diacetic  acid  (see  p.  397).  Start  the  air  current  in  the  usual 
way  and  permit  it  to  run  25  minutes  without  the  application  of 
heat  to  the  urine  under  examination.  Under  these  conditions  the 
preformed  acetone  present  in  the  solution  is  all  removed  (see  p. 
398).  Immediately  attach  a  freshly  prepared  absorption  bottle  or 
introduce  fresh  alkaline  hypoiodite  solution  into  the  original  bottle. 
Apply  heat  to  the  large  test-tube  as  already  described  (see  p.  398  >. 
in  order  to  convert  the  diacetic  acid  into  acetone,  permit  the  air 
current  to  continue  for  the  usual  2^  minute  period,  and  determine 
the  diacetic  acid  value  in  terms  of  acetone  by  the  usual  titration 

procedure  (seep.  399). 

2.  Folin-Messinger-Huppert  Method.  —  Determine  the  com- 
bined acetone  and  diacetic  acid,  in  terms  >>(  acetone,  by  the  Mes- 
singer-Huppert method  (see  p.  390^  and  subsequently  determine  the 

27 


402  PHYSIOLOGICAL    CHEMISTRY. 

acetone  by  Folin's  method  (see  p.  400).  Subtract  the  value  deter- 
mined by  the  second  method  from  that  obtained  in  the  first  method 
to  secure  data  regarding  the  diacetic  acid  content  of  the  urine,  in 
terms  of  acetone. 

XVII.     /?-Oxybutyric  Acid. 

1.  Shaffer's  Method. — Introduce  25-250  c.c.  of  urine1  into  a 
500  c.c.  volumetric  flask  and  add  an  excess  of  basic  lead  acetate  and 
10  c.c.  of  concentrated  ammonium  hydroxide.  Dilute  the  mixture 
to  the  500  c.c.  mark,  shake  the  flask  thoroughly  and  filter.  Transfer 
200  c.c.  of  the  filtrate  to  an  800  c.c.  Kjeldahl  distilling  flask,  add 
300-400  c.c.  of  water,  15  c.c.  of  concentrated  sulphuric  acid  and  a 
little  talcum  and  distil  the  mixture  until  200  to  250  c.c.  of  distillate 
has  been  collected  (A).2  To  this  distillate  (A),  which  contains 
acetone  (both  preformed  and  that  produced  from  diacetic  acid), 
and  volatile  fatty  acids  is  added  5  c.c.  of  10  per  cent  potassium 
hydroxide  and  the  distillate  redistilled  in  order  to  remove  the  vola- 
tile fatty  acids.3  This  second  distillate  (A2)  is  then  titrated  with 
standard  iodine  and  thiosulphate  (see  p.  399).  The  urine-sulphuric 
acid  residue  from  which  distillate  A  was  obtained,  is  again  distilled, 
400-600  c.c.  of  a  0.1-0.5  per  cent  potassium  bichromate  solution 
being  added,  by  means  of  the  dropping  tube,  during  the  process  of 
distillation.4  In  adding  the  bichromate,  care  should  be  taken  not 
to  add  it  faster  than  the  distillate  collects  except  in  cases  where 
the  boiling  fluid  assumes  a  pure  green  color,  thus  indicating  that 
the  bichromate  is  being  used  up  more  rapidly.  After  about  500 
c.c.  of  distillate  (B)  has  collected  20  c.c.  of  a  3  per  cent  solution 
of  hydrogen  peroxide  and  a  few  cubic  centimeters  of  potassium 

1  The  amount  used  depends  upon  the  expected  yield  of  /3-oxybutyric  acid.  In 
the  case  of  urines  which  give  a  strong  ferric  chloride  reaction  for  diacetic  acid, 
or  when  5-10  grams  or  more  of  /3-oxybutyric  acid  is  expected,  it  is  unnecessary 
to  use  more  than  25-50  c.c.  of  urine.  However,  in  case  only  a  trace  of 
/3-oxybutyric  acid  is  expected,  the  volume  should  be  much  larger  as  indicated. 
Under  all  conditions,  the  amount  specified  is  sufficient  for  duplicate  determina- 
tions. It  is  desirable  to  use  such  a  volume  of  urine  as  -contains  the  proper 
amount  of  /3-oxybutyric  acid  to  yield  25-50  milligrams  of  acetone. 

2  This  distilling  flask  should  be  provided  with  a  dropping  tube,  by  means  of 
which  water  may  be  introduced  in  order  to  prevent  the  contents  of  the  flask 
from  becoming  less  than  400  c.c.  in  volume.  Care  should  be  taken  to  use  a 
good  condenser  in  the  distillation,  but  it  is  not  necessary  to  cool  the  distillate 
with  ice. 

3  Formic  acid  is  one  of  the  most  troublesome. 

4  Generally  the  addition  Of  0.5  gram  of  potassium  bichromate  is  sufficient.  In 
case  the  urine  contains  a  high  concentration  of  sugar  or  when  a  large  volume 
of  urine  is  used,  it  may  be  necessary  to  use  2-3  grams  of  the  bichromate. 


urine:  quantitative  analysi  403 

hydroxide  solution  are  added  and  the  mixture  (B)  subjected  to 
redistillation.  Distil  off  about  300  c.c.  and  titrate  this  distillate 
(B2)  as  usual  with  iodine  and  thiosulphate  (see  p.  39 

Calculation. —  The  author  advises  the  use  of  solutions  of  thio- 
sulphate and  iodine  which  are  a  trifle  stronger  than  ,s,,,  1.  .-.,  [03 
Each  cubic  centimeter  of  an  iodine  solution  of  this  strength  is 
equivalent  to  one  milligram  of  acetone  or  to  1704  milligram  of 
j8-oxybutyric  acid.  The  thiosulphate  solution  1-  accepted  as  the 
standard  and  should  he  restandardi/.ed.  Erom  time  to  time,  by  a 
~  solution  of  potassium  hi-iodate. 

2.  Black's  Method. — Render  50  c.c.  of  the  urine  under  examina- 
tion, faintly  alkaline  with  sodium  carbonate  and  evaporate  to  one- 
third  the  original  volume.  Concentrate  to  about  10  c.c.  on  a  water- 
bath,  cool  the  residue,  acidify  it  with  a  few  drops  of  concentrated 
hydrochloric  acid1  and  add  plaster  of  Paris  to  form  a  thick  paste. 
Permit  the  mixture  to  stand  until  it  begins  to  "  set,"  then  break  it  up 
with  a  stout  glass  rod  having  a  blunt  end  and  reduce  the  material  to 
the  consistency  of  a  fairly  dry  coarse  meal.-  Transfer  the  meal  to  a 
Soxhlet  apparatus  and  extract  with  ether  for  two  hours.  At  the  end 
of  this  period  evaporate  the  ether-extract  either  spontaneously  or  in 
an  air  current.  Dissolve  the  residue  in  water,  add  a  little  bone  black, 
if  necessary,  filter  until  a  clear  solution  is  obtained  and  make  up 
the  filtrate  to  a  known  volume  (25  c.c.  or  less)  with  water.  The 
/?-oxybutyric  acid  should  then  he  determined  by  means  of  the 
polariscope. 

3.  Darmstadter's  Method. — This  method  is  based  on  the  fact 
that  crotonic  acid  is  formed  from  /?-oxybutvric  acid  under  the 
influence  of  concentrated  mineral  acids.  The  method  is  as  follows: 
Render  100  c.c.  of  urine  slightly  alkaline  with  sodium  carbonate 
and  evaporate  nearly  to  dryness  on  a  water-hath.  Dissolve  the  resi- 
due in  150-200  c.c.  of  50-55  per  cent  sulphuric  acid,  transfer  the 
acid  solution  to  a  one  liter  distillation  flask  and  connect  it  with  a 
condenser.  Through  the  cork  of  the  flask  introduce  the  stem  of  a 
dropping  funnel  containing  water.  Heat  the  flask  gently  until 
foaming  ceases,  then  use  a  full  flame  and  distil  over  about  300  35" 
c.c.  of  fluid,  keeping  the  volume  <>f  liquid  in  the  distillation  flask 
constant  by  the  addition  of  water  from  the  dropping  funnel  as  the 
distillate  collects.     Ordinarily  it  will  take  about  2   2]  2  hours  to  col- 

1  The  residue  should  give  a  distinct  red  color  with  litmus  paper. 

"Before  this  is  accomplished   it   may,  in   some  cases,  be   accessary   to  add   a 

little  more  plaster  (if   Paris. 


404  PHYSIOLOGICAL    CHEMISTRY. 

lect  this  amount  of  distillate.  Extract  the  distillate  three  times1 
with  ether  in  a  separatory  funnel,  evaporate  the  ether  and  heat 
the  residue  at  1600  C.  for  a  few  minutes  to  remove  volatile  fatty 
acids.  Dissolve  the  residue  in  50  c.c.  of  water,  filter  and  titrate 
this  aqueous  solution  of  crotonic  acid  with  f-Q  sodium  hydroxide 
solution,  using  phenolphthalein  as  indicator. 

Calculation. — One  c.c.  of  y\  sodium  hydroxide  solution  equals 
0.0086  gram  of  crotonic  acid,  1  part  of  crotonic  acid  equals  1.21 
part  of  /?-oxybutyric  acid,  and  1  c.c.  of  j-q  sodium  hydroxide  solu- 
tion equals  0.0 1 041  gram  of  /?-oxybutyric  acid.  To  compute  the 
quantity  of  /?-oxybutyric  acid,  in  grams,  multiply  the  number  of 
cubic  centimeters  of  jq  sodium  hydroxide  solution  used  by  0.01041. 

4.  Bergell's  Method. — Render  100-300  c.c.  of  sugar-free2  urine 
slightly  alkaline  with  sodium  carbonate,  evaporate  the  alkaline  urine 
to  a  syrup  on  a  water-bath,  cool  the  syrup,  rub  it  up  with  syrupy 
phosphoric  acid  (being  careful  to  keep  the  mixture  cool),  20-30 
grams  of  finely  pulverized,  anhydrous  cupric  sulphate  and  20-25 
grams  of  fine  sand.  Mix  the  mass  thoroughly,  place  it  in  a  paper 
extraction  thimble3  and  extract  the  dry  mixture  with  ether  in  a 
Soxhlet  apparatus  (Fig.  125,  page  410).  Evaporate  the  ether,  dis- 
solve the  residue  in  about  25  c.c.  of  water,  decolorize  the  fluid  with 
animal  charcoal, , if  necessary,  and  determine  the  content  of  /?-oxy- 
butyric  acid  by  a  polarization  test. 

5.  Boekelman  and  Bouma's  Method. — Place  25  c.c.  of  urine  in 
a  flask,  add  25  c.c.  of  12  per  cent  sodium  hydroxide  and  25  c.c.  of 
benzoyl  chloride,  stopper  the  flask  and  shake  it  vigorously  for  three 
minutes  under  cold  water.  Remove  the  clear  fluid  by  means  of  a 
pipette,  filter  it  and  subject  it  to  a  polarization  test.  Through  the 
action  of  the  benzoyl  chloride  all  the  lsevo-rotatory  substances  ex- 
cept /?-oxybutyric  acid  will  have  been' removed  and  the  laevo-rotation 
now  exhibited  by  the  urine  will  be  due  entirely  to  that  acid. 

XVIII.     Acidity. 

Folin's  Method. — The  total  acidity  of  urine  may  be  determined 
as  follows :  Place  25  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer  flask 
and  add  15-20  grams  of  finely  pulverized  potassium  oxalate  and 
1-2  drops  of  a   1  per  cent  phenolphthalein  solution  to  the  fluid. 

1  Shaffer  has  recently  called  attention  to  the  fact  that  it  is  extremely  difficult 
to  extract  all  of  the  crotonic  acid  if  but  three  extractions  are  made. 

2  If  sugar  is  present  it  must  be  removed  by  fermentation. 

3  The  Schleicher  and  Schiill  fat-free  extraction  thimble  is  very  satisfactory. 


urine:  quantitative  analysis. 

Shake  the  mixture  vigorously  for   i   2  minutes  and  titrate  if   im- 
mediately with  nr  sodium  hydroxide  until  a  faint  bul  unmistakable 

pink  remains  permanent   on    further   shaking.     Take  the  lunette 
reading- and  calculate  the  acidity  of  the  urine  under  examination. 

Calculation. — If  y  represents  the  number  of  cubic  centimetei 
Yij  sodium  hydroxide  used  and  y'  represents  the  volume  of  urine 
excreted  in  twenty- four  hours,  the  total  acidity  of  the  twenty-four 
hour  urine  specimen  may  be  calculated  by  means  of  the  following 
nroportion : 

25:3- ::y'\x  (acidity  of  24-hour  urine  expressed  in  cubic  centimeters 
of  is  sodium  hydroxide). 

Each  cubic  centimeter  of  f^  sodium  hydroxide  contains  0.004 
gram  of  sodium  hydroxide  and  this  is  equivalent  to  0.0063  grani  "' 
oxalic  acid.  Therefore,  in  order  to  express  the  total  acidity  of  the 
twenty-four  hour  urine  specimen  in  equivalent  grams  of  sodium 
hydroxide,  multiply  the  value  of  x,  as  just  determined,  by  0.004, 
or  multiply  the  value  of  x  by  0.0063  if  it  is  desired  to  express  the 
total  acidity  in  grams  of  oxalic  acid. 

XIX.     Purine  Bases. 

1.  Kriiger  and  Schmidt's  Method. —  This  method  serves  for  the 
determination  of  both  uric  acid  and  the  purine  bases.  The  principle 
involved  is  the  precipitation  of  both  the  uric  acid  and  the  purine 
bases  in  combination  with  copper  oxide  and  the  subsequent  decom- 
position of  this  precipitate  by  means  of  sodium  sulphide.  The  uric 
acid  is  then  precipitated  by  means  of  hydrochloric  acid  and  the 
purine  bases  are  separated  from  the  filtrate  in  the  form  of  their 
copper  or  silver  compounds.  The  nitrogen  content  of  the  precipi- 
tates of  uric  acid  and  purine  bases  is  then  determined  by  means  of 
the  Kjeldahl  method  (see  p.  381)  and  the  corresponding  values  for 
uric  acid  and  purine  bases  calculated.  The  method  is  a-  follows: 
To  400  c.c.  of  albumin-free  urine1  in  a  liter  flask,2  add  24  grams 
of  sodium  acetate,  40  c.c.  of  a  solution  of  sodium  bisulphite8  and 
heat  the  mixture  to  boiling.     Add  40-80  c.c.4  of  a  10  per  cent  solu- 

*If  albumin  is  present,  the  urine  should  be  heated  to  boiling,  acidified  with 
acetic  acid  and  filtered. 

-The  total  volume  of  urine  for  the  twenty-four  hours  should  Ik-  sufficiently 
diluted  with  water  to  make  the  total  volume  of  the  solution  1600-2000  c.c. 

3  A  solution  containing  50  grams  of  Kahlbaum's  commercial  sodium  bisulphite 
in  100  c.c.  of  water. 

*  The  exact  amount  depending  upon  the  contenl  "t"  the  purine  bases 


406  PHYSIOLOGICAL    CHEMISTRY. 

tion  of  cupric  sulphate  and  maintain  the  temperature  of  the  mix- 
ture at  the  boiling-point  for  at  least  three  minutes.  Filter  off  the 
flocculent  precipitate,  wash  it  with  hot  water  until  the  wash  water 
is  colorless,  and  return  the  washed  precipitate  to  the  flask  by  punc- 
turing the  tip  of  the  filter  paper  and  washing  the  precipitate  through 
by  means  of  hot  water.  Add  water  until  the  volume  in  the  flask 
is  approximately  200  c.c.,  heat  the  mixture  to  boiling  and  decom- 
pose the  precipitate  of  copper  oxide  by  the  addition  of  30  c.c.  of 
sodium  sulphide  solution.1  After  decomposition  is  complete,  the 
mixture  should  be  acidified  with  acetic  acid  and  heated  to  boiling 
until  the  separating  sulphur  collects  in  a  mass.  Filter  the  hot  fluid 
by  means  of  a  filter-pump,  wash  with  hot  water,  add  10  c.c.  of  10 
per  cent  hydrochloric  acid  and  evaporate  the  filtrate  in  a  porcelain 
dish  until  the  total  volume  has  been  reduced  to  about  ten  cubic  centi- 
meters. Permit  this  residue  to  stand  about  two  hours  to  allow 
for  the  separation  of  the  uric  acid,  leaving  the  purine  bases  in  solu- 
tion. Filter  off  the  precipitate  of  uric  acid,  using  a  small  filter 
paper,  and  wash  the  uric  acid,  with  water  made  acid  with  sulphuric 
acid,  until  the  total  volume  of  the  original  filtrate  and  the  wash  water 
aggregates  75  c.c.  Determine  the  nitrogen  content  of  the  pre- 
cipitate by  means  of  the  Kjeldahl  method  (see  p.  381),  and  calcu- 
late the  uric  acid  equivalent.2 

Render  the  filtrate  from  the  uric  acid  crystals  alkaline  with 
sodium  hydroxide,  add  acetic  acid  until  faintly  acid  and  heat  to 
700  C.  Now  add  one  cubic  centimeter  of  a  10  per  cent  solution 
of  acetic  acid  and  10  c.c.  of  a  suspension  of  manganese  dioxide3 
to  oxidize  the  traces  of  uric  acid  which  remain  in  the  solution. 
Agitate  the  mixture  for  one  minute,  add  10  c.c.  of  the  sodium  bisul- 
phite solution4  and  5  c.c.  of  a  10  per  cent  solution  of  cupric  sulphate 
and  heat  the  mixture  to  boiling  for  three  minutes.     Filter  off  the 

1  This  is  made  by  saturating  a  one  per  cent  solution*  of  sodium  hydroxide  with 
hydrogen  sulphide  gas  and  adding  an  equal  volume  of  one  per  cent  sodium 
hydroxide. 

Ordinarily  the  addition  of  30  c.c.  of  this  solution  is  sufficient,  but  the  presence 
of  an  excess  of  sulphide  should  be  proven  by  adding  a  drop  of  lead  acetate  to  a 
drop  of  the  solution.  Under  these  conditions  a  dark  brown  color  will  show 
the  presence  of  an  excess  of  sodium  sulphide. 

2  This  may  be  done  by  multiplying  the  nitrogen  value  by  three  and  adding 
three  and  one-half  milligrams  to  the  product  as  a  correction  for  the  uric  acid 
remaining  in  solution  in  the  75  c.c. 

3  Made  by  heating  a  0.5  per  cent  solution  of  potassium  permanganate  with  a 
little  alcohol  until  it  is  decolorized. 

*  To  dissolve  the  excess  of  manganese  dioxide. 


urine:  quantitative  analysi 

precipitate  wash  it  with  hot  water  and  determine  its  nitrogen  con 
tent  by  means  of  the  Kjeldah!  method  I  see  p.  381  >.     [nasmuch  as 
the  composition  and  proportion  of  the  purine  bases  presenl  in  urine 
is  variable,  no  factor  can  be  applied.     The  result  as  regards  these 
bases  must  therefore  be  expressed  in  terms  of  nitrogen. 

2.  Salkowski's  Method. —  Place  400  600  c.c.  of  protein 
urine  in  a  beaker,  [ntroduce  into  another  beaker  30  50  1  ,c.  of  an 
ammoniacal  silver  solution1  with  30  50  c.c.  of  magnesia  mixture,2 
add  some  ammonium  hydroxide  and  if  necessary  some  ammonium 
chloride  to  clear  the  solution.  Nov  add  this  solution  to  the  urine, 
stirring  continually  with  a  glass  rod.  and  allow  the  mixture  to  stand 
for  one-half  hour.  Collect  the  precipitate  on  a  filter  paper,  wash 
it  with  dilute  ammonium  hydroxide  and  finally  wash  it  back  into 
the  original  beaker.  Suspend  the  precipitate  in  600  800  C.C.  of 
water,  add  a  few  drops  of  hydrochloric  acid  and  decompose  it  by 
means  of  hydrogen  sulphide.  Now  heat  the  solution  to  boiling,  bi- 
ter while  hot  and  evaporate  the  filtrate  to  dryness  on  a  water-bath. 
Extract  the  residue  with  20-30  c.c.  of  hot  3  per  cent  sulphuric 
acid  and  allow  the  extract  to  stand  twenty- four  hours.  Filter  off 
the  uric  acid,  wash  it.  make  the  filtrate  ammoniacal  and  precipitate 
the  purine  bases  again  with  silver  nitrate.  Collect  this  precipitate 
on  a  small-sized  chlorine-free  filter  paper,  wash,  dry  .and  incinerate 
it  in  the  usual  manner.  Xow  dissolve  the  ash  in  nitric  acid  and 
titrate  with  ammonium  thiocyanate  according  to  the  Volhard- 
Arnold  method  (see  p.  396).  Calculate  the  content  of  purine  bases 
in  the  urine  examined,  bearing  in  mind  that  in  an  equal  mixture  of 
the  silver  salts  of  the  purine  bases,  such  as  we  have  here,  one  part  of 
silver  corresponds  to  0.277  gram  of  nitrogen  or  to  0.738]  gram 
of  the  bases. 

XX.     Allantoin. 

Paduschka-Underhill-Kleiner  Method. — To  50-100  c.c.  of 
urine  in  a  beaker  add  basic  lead  acetate  until  no  more  precipitate 
forms.  Filter  and  pass  hydrogen  sulphide  gas  through  an  aliquol 
portion  of  the  filtrate  to  remove  the  excess  of  lead.3  Filter  again, 
drive  off  the  hydrogen  sulphide  by  heat  and  treat  an  aliquot  por- 

1  Prepared  by  dissolving  26  gram-  of  silver  nitrate  in  aboul  500  C.C.  of  water, 
adding  enough  ammonium  hydroxide  to  redissolve  the  precipitate  which  forms 
upon  the  first  addition  of  the  ammonia  and  making  the  balance  of  the  mixture 
up  to  1  liter  with  water. 

"Directions  for  preparation  may  be   found  on  page  295. 

'In  the  original  method  of  Paduschka  sodium  sulphate  is  used  for  tins 
purpose. 


408  PHYSIOLOGICAL    CHEMISTRY. 

tion  of  the  filtrate  with  a  10  per  cent  solution  of  silver  nitrate  until 
precipitation  is  complete.1  Filter  off  this  precipitate,  wash  it  with 
water  and  determine  its  nitrogen  content  by  means  of  the  Kjeldahl 
method  (see  p.  381).  This  is  the  "purine  nitrogen."  Render  an 
aliquot  portion  of  the  filtrate  faintly  alkaline,2  with  a  one  per  cent 
solution  of  ammonium  hydroxide  and  add  50—100  c.c.  of  a  10  per 
cent  solution  of  silver  nitrate.  If  allantoin  be  present  a  white,  floc- 
culent  precipitate  will  form  and  gradually  sink  to  the  bottom  of  the 
solution.  Filter,  wash  the  precipitate  free  from  ammonium  hydrox- 
ide by  means  of  a  one  per  cent  solution  of  sodium  sulphate  and 
determine  its  nitrogen  content  by  the  Kjeldahl  method  (see  p.  381). 

XXI.     Oxalic  Acid. 

Salkowski-Autenrieth  and  Barth  Method. — Place  the  twenty- 
four  hour  urine  specimen  in  a  precipitating  jar,  add  an  excess  of 
calcium  chloride,  render  the  urine  strongly  ammoniacal,  stir  it  well 
and  allow  it  to  stand  18-20  hours.  Filter  off  the  precipitate,  wash 
it  with  a  small  amount  of  water  and  dissolve  it  in  about  30  c.c.  of 
a  hot  15  per  cent  solution  of  hydrochloric  acid.  By  means  of  a 
separatory  funnel  extract  the  solution  with  150  c.c.  of  ether  which 
contains  3  per  cent  of  alcohol,  repeating  the  extraction  four  or  five 
times  with  fresh  portions  of  ether.  Unite  the  ethereal  extracts, 
allow  them  to  stand  for  an  hour  in  a  flask  and  then  filter  through 
a  dry  filter  paper.  Add  5  c.c.  of  water  to  the  filtrate,  to  prevent 
the  formation  of  diethyl  oxalate  when  the  solution  is  heated,  and 
distil  off  the  ether.  If  necessary,  decolorize  the  liquid  with  animal 
charcoal  and  filter.  Concentrate  the  filtrate  to  3-5  c.c,  add  a  little 
calcium  chloride  solution,  make  it  ammoniacal  and  after  a  few  min- 
utes render  it  slightly  acid  with  acetic  acid.  Allow  the  acidified 
solution  to  stand  several  hours,  collect  the  precipitate  of  calcium 
oxalate  on  a  washed  filter  paper,3  wash,  incinerate  strongly  (to 
CaO)  and  weigh  in  the  usual  manner. 

Calculation. — Since  56  parts  of  CaO  are  equivalent  to  90  parts 
of  oxalic  acid,  the  quantity  of  oxalic  acid  in  the  volume  of  urine 
taken  may  be  determined  by  multiplying  the  weight  of  CaO  by  the 
factor  1. 607 1. 

XXII.     Total  Solids. 

1.  Drying  Method. — Place  5  c.c.  of  urine  in  a  weighed  shallow 
dish,  acidify  very  slightly  with  acetic  acid  (1-3  drops)  and  dry  it 

1  Ordinarily  from  20-30  c.c.  is  required. 

2  Using  litmus  as  the  indicator. 

3  Schleicher  and  Schull,  No.  589,  is  satisfactory. 


urine:  quantitative  analysis.  409 

in  vacuo  in  the  presence  of  sulphuric  acid,  to  constant   weight. 
Calculate  the  percentage  of  solids  in  the  mine  sample  and  the  total 

solids  for  the  twenty-four  hour  period. 

Practically  all  the  methods  the  technique  of  which  includes  • 
poration  at  an   increased    temperature,   either   under   atmospheric 
conditions  or  in  vacuo  are  attended  with  error. 

2.  Calculation  by  Long's  Coefficient. — The  quantity  of  solid 
material  contained  in  the  urine  excreted  for  any  twenty-four  hour 
period  may  be  approximately  computed  by  multiplying  the  second 
and  third  decimal  figures  of  the  specific  gravity  by  2.6.  This  gives 
us  the  number  of  grams  of  solid  matter  in  one  liter  of  urine  Fr<  >m 
this  value  the  total  solids  for  the  twenty-four  hour  period  may 
easily  be  determined. 

Calculation. — If  the  volume  of  urine  for  the  twenty- four  hours 
was  1 120  c.c.  and  the  specific  gravity  I.018,  the  calculation  would 
be  as   follows : 
(a)  18  X  2.6  =  46.8  grams  of  solid  matter  in  1  liter  of  urine. 

(0\   ij — iS =52.4  grams  of  solid  matter  in   1120  c.c.   of 

1000 

urine. 

Long's  coefficient  was  determined  for  urine  whose  specific  grav- 
ity was  taken  at  25 °  C.  and  is  probably  more  accurate,  for  con- 
ditions obtaining  in  America,  than  the  older  coefficient  of  Haeser, 

2-33- 


CHAPTER    XXIII, 


Fig.  125. 


QUANTITATIVE  ANALYSIS   OF  MILK,   GASTRIC 
JUICE   AND    BLOOD. 

(a)    Quantitative  Analysis  of  Milk. 

1.  Specific  Gravity. — This  may  be  determined  conveniently  by 
means  of  a  Soxhlet,  Veith  or  Ouevenne  lactometer.  A  lactometer 
reading-  of  320  denotes  a  specific  gravity  of  1.032.  The  determin- 
ation should  be  made  at  about  6o°  F. 
and  the  lactometer  reading  corrected 
by  adding  or  subtracting  o.i°  for 
every  degree  F.  above  or  below  that 
temperature. 

2.  Fat. —  (a)  Adams'  Paper  Coil 
Method.— Introduce  about  5  c.c.  of 
milk  into  a  small  beaker,  quickly  ascer- 
tain the  weight  to  centigrams,  stand  a 
fat-free  coil1  in  the  beaker  and  incline 
the  vessel  and  rotate  the  coil  in  order 
to  hasten  the  absorption  of  the  milk. 
Immediately  upon  the  complete  absorp- 
tion of  the  milk  remove  the  coil  and 
again  quickly  ascertain  the  weight  of 
the  beaker.  The  difference  in  the 
weights  of  the  beaker  at  the  two 
weighings  represents  the  quantity  of 
milk  absorbed  by  the  coil.  Dry  the 
coil  carefully  at  a  temperature  below 
ioo°  C.  and  extract  it  with  ether  for 
3-5  hours  in  a  Soxhlet  apparatus 
(Fig.  125,  p.  410),  using  a  safety 
water-bath,  heat  the  flask  containing 
the  fat  to  constant  weight  at  a  tem- 
perature below  ioo°  C. 

Calculation. — Divide  the  weight  of 
fat,  in  grams,  by  the  weight  of  milk,  in  grams.  The  quotient  is  the 
percentage  of  fat  contained  in  the  milk  examined. 

1  Very  satisfactory  coils  are  manufactured  by  Schleicher  and  Schiill. 

410 


Soxhlet  Apparatus. 


QUANTITATIVE    ANALYSIS    "I      MILK. 


4!I 


fK 


Fes  i 
Lactosi  OP1  . 


(b)  Approximate  Determination  by  l:eser's  LactOSCOpe.  Milk 
is  opaque  mainly  because  of  the  suspended  fal  globules  and  there- 
fore by  means  of  the  estimation  oi  this  opacitj  we  may  obtain  data 

as  to  the  approximate  content  of  Fat.  Feser's  lacto- 
scope  (Fig.  126,  p.  41  1  )  may  be  used  for  this  purpose. 
Proceed  as  follows:  By  means  of  the  graduated  pip- 
ette accompanying  the  instrument  introduce  4  c.e. 
of  milk  into  the  lactoscope.  Add  water  gradually, 
shaking  after  each  addition,  and  note  the  point  ai 
which  the  black  lines  upon  the  inner  white  glass  cylin- 
der are  distinctly  visible.  Observe  the  point  on  the 
graduated  scale  of  the  lactoscope  which  is  level  with 
the  surface  of  the  diluted  milk.  This  reading  repre- 
sents the  percentage  of  fal  present  in  the  undiluted 
milk.  Pure  milk  should  contain  at  least  3  per  cent 
of  fat. 

3.  Total  Solids.1 — Introduce  2-5  grams  of  milk 
into  a  weighed  flat-bottomed  platinum  dish-  and 
quickly  ascertain  the  weight  to  milligrams.     Expel  the 

major  portion  of  the  water  by  heating  the  open  dish  on  a  water-bath 
and  continue  the  heating  in  an  air-bath  or  water  oven  at  qj^-ioo0 
C.  until  the  weight  is  constant.  (If  platinum  dishes  are  employed 
this  residue  may  be  used  in  the  determination  of  ash  according  to 
the  method  described  below.) 

Calculation. — Divide  the  weight  of  the  residue,  in  grams,  by  the 
weight  of  milk  used,  in  grams.     The  quotient  is  the  percental 
solids  contained  in  the  milk  examined. 

4.  Ash. — Heat  the  dry  solids  from  2-$  grams  of  milk,  obtained 
according  to  the  method  just  given,  over  a  very  low  flame8  until 
a  white  or  light  gray  ash  is  obtained.  Cool  the  dish  in  a  desiccator 
and  weigh.  (This  ash  may  be  used  in  testing  for  preservative-  ac- 
cording to  directions  on  page  226.) 

1  The  percentage  of  total  solids  may  be  calculated  from  the  specific  gravity  and 
percentage  of  fat  by  means  of  the  following   formula  winch  has  been  pro 
by  Richmond  : 

S  =  0.25    L  +  1.2    F  +  o.  14 
S  =  total  solids. 
L  =  lactometer  reading. 
F  =  fat  content. 

'Lead  foil  dishes,  costing  only  aboul  one  dollar  per  gross,  make  a  very 
satisfactory  substitute  for  the  platinum  dishes. 

3  Great  care  should  be  used  in  this  ignition,  the  dish  ai  no  time  being  heated 
above   a  faint  redness,  as  chlorides   may   volatilize. 


412  PHYSIOLOGICAL    CHEMISTRY. 

5.  Proteins. — Introduce  a  known  weight  of  milk  (5-10  grams) 
into  a  500  c.c.  Kjeldahl  digestion  flask  and  add  20  c.c.  of  con- 
centrated sulphuric  acid  and  about  0.2  gram  of  cupric  sulphate. 
Expel  the  major  portion  of  the  water  by  heating  over  a  low  flame 
and  finally  use  a  full  flame  and  allow  the  mixture  to  boil  1-2  hours. 
Complete  the  determination  according  to  the  directions  given  under 
Kjeldahl  Method,  page  381. 

Calculation — Multiply  the  total  nitrogen  content  by  the  factor 
6.371  to  obtain  the  protein  content  of  the  milk  examined. 

6.  Caseinogen. — Mix  about  20  grams  of  milk  with  40  c.c.  of  a 
saturated  solution  of  magnesium  sulphate  and  add  the  salt  in  sub- 
stance until  no  more  will  dissolve.  The  precipitate  consists  of 
caseinogen  admixed  with  a  little  fat  and  lacto-globulin.  Filter  off 
the  precipitate,  wash  it  thoroughly  with  a  saturated  solution  of 
magnesium  sulphate,2  transfer  the  filter  paper  and  precipitate  to 
a  Kjeldahl  digestion  flask  and  determine  the  nitrogen  content  ac- 
cording to  the  directions  given  in  the  previous  experiment. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to 
obtain  the  casein  content. 

7.  Lactalbumin. — To  the  filtrate  and  washings  from  the  deter- 
mination of  caseinogen,  as  just  explained,  add  Almen's  reagent3 
until  no  more  precipitate  forms.  Filter  off  the  precipitate  and 
determine  the  nitrogen  content  according  to  the  directions  given 
under  Proteins,  above. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to  ob- 
tain the  lactalbumin  content. 

8.  Lactose. — To  about  350  c.c.  of  water  in  a  beaker  add  20 
grams  of  milk,  mix  thoroughly,  acidify  the  fluid  with  about  2  c.c. 
of  10  per  cent  acetic  acid  and  stir  the  acidified  mixture  continuously 
until  a  flocculent  precipitate  forms.  At  this  point  the  reaction 
should  be  distinctly  acid  to  litmus.  Heat  the  solution  to  boiling 
for  one-half  hour,  filter,  rinse  the  beaker  thoroughly  and  wash  the 
precipitated  proteins  and  the  adherent  fat  with  .hot  water.  Com- 
bine the  filtrate  and  wash  water  and  concentrate  the  mixture  to  about 

* 

1  The  usual  factor  employed  for  the  calculation  of  protein  from  the  nitrogen 
content  is  6.25  and  is  based  on  the  assumption  that  proteins  contain  on  the 
average  16  per  cent  of  nitrogen.  This  special  factor  of  6.37  is  used  here  to 
calculate  the  protein  content  from  the  total  nitrogen,  since  the  principal  protein 
constituents  of  milk,  i.  e.,  caseinogen  and  lactalbumin,  contain  15.7  per  cent 
of  nitrogen. 

2  Preserve  the  filtrate  and  washings  for  the  determination  of  lactalbumin. 

3  Almen's  reagent  may  be  prepared  by  dissolving  5  grams  of  tannin  in  240  c.c. 
of  50  per  cent  alcohol  and  adding  10  c.c.  of  25  per  cent  acetic  acid. 


QUANTITATIVE    ANALYSIS    OF    MILK.  413 

150  c.c.     Cool  the  solution  and  dilute  it  to  200  c.c.  in  a  volumetric 
flask.     Titrate  this  sugar  solution  according  to  directions   - 

under  Fehling's  Method,  page  367. 

Calculation.     Make  1 1  u-  calculation  according  to  din  given 

under  Fehling's  Method,  p.  307,  bearing  in  mind  that   to  < 
Fehling's  solution  is  completely  reduced  by  O.06/6  gram  of  lactose. 

(b)    Quantitative  Analysis  of  Gastric  Juice. 
Topfer's  Method. 

This  method  is  much  less  elaborate  than  many  others  but  is  suffi- 
ciently accurate  for  ordinary  clinical  purposes.  The  method  em- 
braces the  volumetric  determination  of  (1)  total  acidity,  |  2  1  com- 
bined acidity,  and  (3)  free  acidity,  and  the  subsequent  calculation 
of  (4)  acidity  due  to  organic  acids  and  acid  salts,  from  the  data 
thus  obtained. 

Strain  the  gastric  contents  and  introduce  10  c.c.  of  the  strained 
material  into  each  of  three  small  beakers  or  porcelain  dishes.1 
Label  the  vessels  A,  B  and  C,  respectively,  and  proceed  with  the 
analysis  according  to  the  directions  given  below. 

1.  Total  Acidity.2 — Add  3  drops  of  a  1  per  cent  alcoholic  solu- 
tion of  phenolphthalein3  to  the  contents  of  vessel  A  and  titrate  with 
yg-  sodium  hydroxide  solution  until  a  dark  pink  color  is  produced 
which  cannot  be  deepened  by  further  addition  of  a  drop  of  -fj 
sodium  hydroxide.  Take  the  burette  reading  and  calculate  the 
total  acidity. 

Calculation. — The  total  acidity  may  be  expressed  in  the  following 
ways : 

1.  The  number  of  cubic  centimeters  of  yu  sodium  hydroxide  solu- 
tion necessary  to  neutralize  100  c.c.  of  gastric  juice. 

2.  The  weight  (in  grams)  of  sodium  hydroxide  necessary  to 
neutralize  100  c.c.  of  gastric  juice. 

3.  The  weight  (in  grams)  of  hydrochloric  acid  which  the  total 
acidity  of  100  c.c.  of  gastric  juice  represents,  i.  c,  percentage  of  hy- 
drochloric acid. 

The  forms  of  expression  most  frequently  employed  are  1  and  3, 
preference  being  given  to  the  former. 

'If  sufficient  gastric  juice  is  not  available  it  may  be  diluted  with  water  or  a 
smaller  amount,  e.  g.,  5  c.c,  taken  for  each  determination. 
"  This  includes  free  and  combined  acid  and  acid  salts. 
3  One  gram  of  phenolphthalein  dissolved  in  ioo  c.c.  of  05  per  cent  alcohol. 


4 14  PHYSIOLOGICAL    CHEMISTRY. 

In  making  the  calculation  note  the  number  of  cubic  centimeters 
of  yq  sodium  hydroxide  required  to  neutralize  10  c.c.  of  the  gastric 
juice  and  multiply  it  by  10  to  obtain  the  number  of  cubic  centimeters 
necessary  to  neutralize  100  c.c.  of  the  fluid.  If  it  is  desired  to  ex- 
press the  acidity  of  ioo  c.c.  of  gastric  juice  in  terms  of  hydro- 
chloric acid,  by  weight,  multiply  the  value  just  obtained  by  0.00365.1 

2.  Combined  Acidity.2 — Add  3  drops  of  sodium  alizarin  sul- 
phonate  solution3  to  the  contents  of  vessel  B  and  titrate  with 
•^  sodium  hydroxide  solution  until  a  violet  color  is  produced.  In 
this  titration  the  red  color,  which  appears  after  the  tinge  of  yellow 
due  to  the  addition  of  the  indicator  has  disappeared,  must  be  en- 
tirely replaced  by  a  distinct  violet  color.  Take  the  burette  reading 
and  calculate  the  combined  acidity. 

Calculation. — Since  the  indicator  used  reacts  to  all  acidities  except 
combined  "acidity  in  order  to  determine  the  number  of  cubic  centi- 
meters of  -yq  sodium  hydroxide  necessary  to  neutralize  the  combined 
acidity  of  10  c.c.  of  the  gastric  juice,  we  must  subtract  the  burette 
reading  just  obtained  from  the  burette  reading  obtained  in  the  de- 
termination of  the  total  acidity.  The  data  for  100  c.c.  of  g"astric 
juice  may  be  calculated  according  to  the  directions  given  under 
Total  Acidity,  page  413. 

3.  Free  Acidity.4 — Add  4  drops  of  di-methyl-amino-azobenzene 
(Topfer's  reagent)  solution5  to  the  contents  of  the  vessel  C  and 
titrate  with  -3^  sodium  hydroxide  solution  until  the  initial  red  color 
is  replaced  by  lemon  yellow.®  Take  the  burette  reading  and  calcu- 
late the  free  acidity. 

Calculation. — The  indicator  used  reacts  only  to  free  acid,  hence 
the  number  of  cubic  centimeters  of  -^  sodium  hydroxide  used  indi- 
cates the  volume  necessary  to  neutralize  the  free  acidity  of  10  c.c. 
of  gastric  juice.  To  determine  the  data  for  100  c.c.  of  gastric 
juice  proceed  according  to  the  directions  given  under  Total  Acidity, 
page  413. 

4.  Acidity  due  to  Organic  Acids  and  Acid  Salts. — This  value 
may  be  conveniently  calculated  by  subtracting  the  number  of  cubic 

*One  c.c.  of  **  hydrochloric  acid  contains  0.00365  gram  of  hydrochloric  acid. 

2  Hydrochloric  acid   combined   with   protein   material. 

3  One  gram  of  sodium  alizarin  sulphonate  dissolved  in   100  c.c.  of  water. 

4  Hydrochloric  acid  not  combined  with  protein  material. 

5  One-half  gram  dissolved  in  100  c.c.  of  95  per  cent  alcohol. 

6  If  the  lemon  yellow  color  appears  as  soon  as  the  indicator  is  added  it  denotes 
the  absence  of  free  acid. 


QUANTITATIVE    ANALYSIS    OF    GASTRK      M   Ml.  415 

centimeters  of  ,N()  sodium  hydroxide  used  in  neutralizing  the  contents 
of  vessel  C  from  the  number  of  cubic  centimeters  of  ,s,,  sodium 
hydroxide  solution  used  in  neutralizing  the  contents  of  vessel  B. 
The  remainder  indicates  the  number  of  cubic  centimeters  of  }s„ 
sodium  hydroxide  solution  necessary  to  neutralize  the  acidity  due 
to  organic  acids  and  acid  salts  present  in  [0  c.c.  of  gastric  juice. 
The  data  for  100  c.c.  of  gastric  juice  may  be  calculated  according 
to  directions  given  under  Total  Acidity,  page  413. 

(c)    Quantitative  Analysis  of  Blood. 

For  the  methods  involved  in  the  quantitative  examination  of  blood 
see  Chapter  XII. 


APPENDIX. 

Almen's  Reagent.1 — Dissolve  5  grams  of  tannin  in  240  c.c.  of 
50  per  cent  alcohol  and  add  10  c.c.  of  25  per  cent  acetic  acid. 

Ammoniacal  Silver  Solution.2 — Dissolve  26  grams  of  silver 
nitrate  in  about  500  c.c.  of  water,  add  enough  ammonium  hydroxide 
to  redissolve  the  precipitate  which  forms  upon  the  first  addition  of 
the  ammonium  hydroxide  and  make  the  volume  of  the  mixture  up  to 
1  liter  with  water. 

Arnold-Lipliawsky  Reagent.3 — This  reagent  consists  of  two 
definite  solutions  which  are  ordinarily  preserved  separately  and 
mixed  just  before  using.  The  two  solutions  are  prepared  as 
follows : 

(a)  One  per  cent  aqueous  solution  of  potassium  nitrite. 

(b)  One  gram  of  p-amino-acetophenon  dissolved  in  100  c.c.  of 
distilled  water  and  enough  hydrochloric  acid  (about  2  c.c.)  added 
drop  by  drop,  to  cause  the  solution,  which  is  at  first  yellow,  to 
become  entirely  colorless.     An  excess  of  acid  must  be  avoided. 

Barfoed's  Solution.4 — Dissolve  4.5  grams  of  neutral,  crystal- 
lized cupric  acetate  in  100  c.c.  of  water  and  add  0.12  c.c.  of  50  per 
cent  acetic  acid. 

Baryta  Mixture.5 — A  mixture  consisting  of  one  volume  of  a 
saturated  solution  of  barium  nitrate  and  two  volumes  of  a  saturated 
solution  of  barium  hydroxide. 

Benedict's  Solutions.6 — First  Modification. — Benedict's  modi- 
fied Fehling  solution  consists  of  two  definite  solutions — a  cupric 
sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be 
prepared  as  follows : 

Cupric  sulphate  solution  =  34.65  grams  of  cupric  sulphate  dis- 
solved in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  100  grams  of  anhydrous  sodium  car- 

1  Ott's  precipitation  test,  p.  321.      Determination  of  lactalbumin,  p.  412. 

2  Salkowski's  method,  page  407. 

3  Arnold-Lipliawsky  reaction,  page  331. 

4  Barfoed's  test,  pages  31  and  313. 

6  Isolation  of  urea  from  urine,  page  269. 

6  Benedict's  modifications  of  Fehling' s  test,  pages  28  and  309,  and  Benedict's 
Method,  page  368. 

416 


APPENDIX.  ;  i  — 

bonate  and  173  grams  of  Rochelle  salt  dissolved  in  water  and  made 
up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stop- 
pered bottles  and  mixed  in  equal  volumes  when  needed   for  use. 

This  is  done  to  prevent  deterioration. 

Second  Modification. — Very  recently  Benedicl  has  further  modi- 
fied his  solution  and  has  succeeded  in  obtaining  one  which  does  not 
deteriorate  upon  long-  standing.     It  has  the  following  composition: 

Cupric    sulphate    17.3  grams. 

Sodium   citrate    i~.VO  grams. 

Sodium    carbonate    100.0 

Distilled   water  to   make    1    liter. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate 
in  about  600  c.c.  of  water.  Pour  (through  a  folded  filter  paper  if 
necessary)  into  a  glass  graduate  and  make  up  to  850  c.c.  1  dissolve 
the  cupric  sulphate  in  about  100  c.c.  of  water  and  make  up  to  [50 
c.c.  Pour  the  carbonate-citrate  solution  into  a  large  beakei 
casserole  and  add  the  cupric  sulphate  solution  slowly,  with  constant 
stirring.  The  mixed  solution  is  ready  for  use  and  does  not  dete- 
riorate upon  long  standing. 

Benedict's  solution  as  used  in  the  quantitative  determination  ol 
sugar  consists  of  three  separate  solutions,  the  two  mentioned  under 
First  Modification  and  in  addition  a  potassium  ferro-thioeyaiiale 
solution.  This  third  solution  contains  15  grams  of  potassium  fer- 
rocyanide,  62.5  grams  of  potassium  thiocyanate  and  50  gram-  <u 
anhydrous  sodium  carbonate  dissolved  in  water  ami  made  up  t"  500 
c.c.  In  preparing  the  Benedict's  solution  for  quantitative  work  the 
three  solutions  mentioned  are  combined  in  equal  parts. 

Black's  Reagent.1 — Made  by  dissolving  5  gram-  of  ferric  chlor- 
ide and  0.4  gram  of  ferrous  chloride  in  100  c.c.  of  water. 

Boas'  Reagent.2 — Dissolve  5  grams  of  resorcin  and  3  grams  of 
sucrose  in  100  c.c.  of  95  per  cent  alcohol. 

Congo  Red.0 — Dissolve  0.5  gram  of  congo  red  in  00  c.c.  of 
water  and  add  10  c.c.  of  95  per  cent  alcohol. 

Ehrlich's  Diazo  Reagent.4 — Two  separate  solution-  should  be 
prepared  and  mixed  in  definite  proportions  when  needed  for  use. 

(a)    Five  grams  of  sodium  nitrite  dissolved  in  1  liter  of  distilled 

water. 

1  Black's  reaction,  page  332. 
-Test  for  free  acid,  page  124. 
3  Test  for  free  acid,  page  124. 
'  Ehrlich's   diazo   reaction,   page  342. 


41 8  PHYSIOLOGICAL    CHEMISTRY.' 

(b)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric 
acid  in  1  liter  of  distilled  water. 

Solutions  a  and  b  should  be  preserved  in  well  stoppered  vessels 
and  mixed  in  the  proportion  1  :  50  when  required.  Green  asserts 
that  greater  delicacy  is  secured  by  mixing  the  solutions  in  the  pro- 
portion 1 :  100.  The  sodium  nitrite  deteriorates  upon  standing 
and  becomes  unfit  for  use  in  the  course  of  a  few  weeks, 

Esbach's  Reagent.1 — Dissolve  10  grams  of  picric  acid  and  20 
grams  of  citric  acid  in  1  liter  of  water. 

Fehling's  Solution.2 — Fehling's  solution  is  composed  of  two 
definite  solutions — a  cupric  sulphate  solution  and  an  alkaline  tartrate 
solution,  which  may  be  prepared  as  follows  : 

Cupric  sulphate  solution  ==  34.65  grams  of  cupric  sulphate  dis- 
solved in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =125  grams  of  potassium  hydroxide 
and  173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to 
500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stop- 
pered bottles  and  mixed  in  equal  volumes  when  needed  for  use. 
This  is  done  to  prevent  deterioration. 

Ferric  Alum  Solution.3 — A  cold  saturated  solution. 

Folin-Shaffer  Reagent.4 — This  reagent  consists  of  500  grams 
of  ammonium  sulphate,  5  grams  of  uranium  acetate  and  60  c.c.  of 
10  per  cent  acetic  acid  in  650  c.c.  of  distilled  water. 

Furfurol  Solution.5 — Add  1  c.c.  of  furfurol  to  1000  c.c.  of  dis- 
tilled water. 

Gallic  Acid  Solution.6 — A  saturated  alcoholic  solution. 

Guaiac  Solution.7 — Dissolve  0.5  gram  of  guaiac  resin  in  30  c.c. 
of  95  per  cent  alcohol. 

Giinzberg's  Reagent.8 — Dissolve  2  grams  of  phloroglucin  and  1 
gram  of  vanillin  in  100  c.c.  of  95  per  cent  alcohol. 

Hammarsten's  Reagent.9 — Mix  1  volume  of  25  per  cent  nitric 
acid  and   19  volumes  of  25  per  cent  hydrochloric  acid  and  add 

1  Esbach's  method,  page  366. 

2  Fehling's  method,  page  367.      Fehling's  test,  pages  27  and  308. 

3  Volhard-Arnold  method,   page  396. 

4  Folin-Shaffer  method,  page  372. 

5  Mylius's  modification  of  Pettenkofer's  test,  pages  156  and  326.  v.  Udransky's 
test,  pages  156  and  326. 

G  Gallic  acid  test,  page  225. 

7  Guaiac  test,  pages  178,   196  and  322. 

8  Test  for  free  acid,  page  123. 

0  Hammarsten's  reaction,  pages  155  and  325. 


APPENDIX.  419 

i  volume  of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol. 
It  is  preferable  thai  the  acid  mixture  be  prepared  in  advance  and 

allowed  to  stand  until  ycll< i\\  in  o ill ir  bef<  ire  adding  it  t<  1  the  alc< »hol. 

Hopkins-Cole  Reagent.1-  To  one  liter  of  a  saturated  olution 
of  oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  1  Ik- 
mixture  to  stand  until  the  evolution  of  gas  ceases.  Filter  and 
dilute  with  2  3  volumes  of  water. 

Hypobromite  Solution.- — The  ingredients  of  this  solution 
should  be  prepared  in  the  form  of  tzvo  separate  solutions  which  may 
be  united  as  needed. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  [25 
grams  of  bromine  and  make  the  total  volume  of  the  solution  1 
liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  -pen lie  gravity  of 
1.25.     This  is  approximately  a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles  and  when 
needed  for  use  mix  equal  volumes  of  solution  a,  solution  />.  and 
water. 

Iodine  Solution/' — Prepare  a  2  per  cent  solution  of  potassium 
iodide  and  add  sufficient  iodine  to  color  it  a  deep  yellow. 

Jolles'  Reagent.4 — This  reagent  has  the  following  composition: 

Succinic  acid 40  grams. 

Mercuric  chloride   20  grains. 

Sodium  chloride    20  grams. 

Distilled    water     1000  gram-. 

Kraut's  Reagent.-"' — Dissolve  2j2  grams  of  potassium  iodide 
in  water  and  add  80  grams  of  bismuth  subnftrate  dissolved  in  200 
grams  of  nitric  acid  (sp.  gr.  1.1S).  Permit  the  potassium  nitrate 
to  crystallize  out,  then  filter  it  off  and  make  the  filtrate  up  to  1 
liter   with   water. 

Lugol's  Solution.0 — Dissolve  4  gram-  of  iodine  and  6  gram-  ol 
potassium  iodide  in  100  c.c.  of  distilled  water. 

Magnesia  Mixture.7 — Dissolve  175  grams  of  magnesium  sul- 
phate and  350  grams  of  ammonium  chloride  in  14m)  c.c.  of  distilled 

1  Hopkins-Cole   reaction,  page  91. 
J  Methods  for  determination  of  urea,  page  374. 
'■'  Iodine  test,  page  44. 
'Jolles'  reaction,  pages  98  and  316. 
'Rosenheim's  bismuth  test    for  choline,   page   252. 
"Gunning's  iodoform  test,  page  328,  and  Bardach's  reaction  pagi 
'Sodium  hydroxide   and   potassium   nitrate    fusion   method    for   detennii 
of  total  phosphorus,  page  390. 


420  PHYSIOLOGICAL    CHEMISTRY. 

water.  Add  700  grams  of  concentrated  ammonium  hydroxide, 
mix  thoroughly  and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

Millon's  Reagent.1 — Digest  1  part  (by  weight)  of  mercury  with 
2  parts  (by  weight)  of  nitric  acid  (sp.  gr.  1.42)  and  dilute  the  re- 
sulting solution  with  2  volumes  of  water. 

Molisch's  Reagent.2 — A  15  per  cent  alcoholic  solution  of 
a-naphthol. 

Molybdic  Solution.3 — Molybdic  solution  is  prepared  as  follows, 
the  parts  being  by  weight: 

Molybdic  acid 1  part. 

Ammonium  hydroxide  (sp.  gr.  0.96) 4  parts. 

Nitric  acid    (sp.  gr.   1.2) 15  parts. 

Moreigne's  Reagent.4 — Combine  20  grams  of  sodium  tungstate, 
10  grams  of  phosphoric  acid  (sp.  gr.  1.13)  and  100  c.c.  of  water. 
Boil  the  mixture  for  twenty  minutes,  add  water  to  make  the  vol- 
ume of  the  solution  equivalent  to  the  original  volume  and  acidify 
with  hydrochloric  acid. 

■  Morner's  Reagent.5 — Thoroughly  mix  1  volume  of  formalin,  45 
volumes  of  distilled  water  and  55  volumes  of  concentrated  sulphuric 
acid. 

Nakayama's-  Reagent.6 — Prepared,  by  combining  99  c.c.  of  al- 
cohol and  1  c.c.  of  fuming  hydrochloric  acid  containing  4  grams 
of  ferric  chloride  per  liter. 

Neutral  Olive  Oil.7 — Shake  ordinary  olive  oil  with  a  10  per 
cent  solution  of  sodium  carbonate,  extract  the  mixture  with  ether 
and  remove  the  ether  by  evaporation.  The  residue  is  neutral  olive 
oil. 

Nylander's  Reagent.8 — Digest  2  grams  of  bismuth  subnitrate 
and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  solution 
of  potassium  hydroxide.  The  reagent  should  then  be  cooled  and 
filtered. 

1  Millon's  reaction,  page  90. 

2  Molisch's  reaction,  page  23. 

3  Sodium  hydroxide  and  potassium  nitrate  fusion  method  for  determination 
of  total  phosphorus,  page  390. 

4  Moreigne's  reaction,  page  275. 

5  Morner's  test,  page  84. 

s  Nakayama's  reaction,  pages   155  and  324. 
'  Emulsification  of  fats,  page  136. 

6  Nylander's  test,  pages  30  and  312. 


\ri'i:.\Dix. 

Obermayer's  Reagent.1  Add  _•  .\  grams  of  ferric  chloride  to 
a  liter  of  hydrochloric  acid   (sp.  gr.   [.19). 

Oxalated  Plasma. 2 — Allov*  arterial  blood  to  run  into  an  equal 
volume  of  0.2  per  rent  ammonium  oxalate  solution, 

Para-dimethylaminobenzaldehyde  Solution.1  This  solution  1- 
made  by  dissolving  5  grains  of  para-dimcthvlaminobenzaldehyde 
in  100  c.c.  of  10  per  cent  sulphuric  acid. 

Para-phenelenediamine  Hydrochloride  Solution.'  I  wo  grams 
dissolved  in   too  c.c.  of  water. 

Phenolphthalein/'  -Dissolve  1  gram  of  phenolphthalein  in  [00 
c.c.  of  95  per  cent  alcohol. 

Phenylhydrazine  Mixture.1'' — This  mixture  is  prepared  by  com- 
bining 1  part  of  phenylhydrazine-hydrochloride  and  2  parts  of  sod- 
ium acetate  by  zveight.     These  are  thoroughly  mixed  in  a  mortar. 

Phenylhydrazine-Acetate  Solution.7 — This  solution  is  prepared 
by  mixing'  1  volume  of  glacial  acetic  acid.  1  volume  <>t  water  and 
2   volumes  of  phenylhydrazine    (the  base). 

Purdy's  Solution.8 — Purdy's  solution  has  the  following  com- 
position : 

Cupric  sulphate  4-75-'  grams. 

Potassium   hydroxide    235       gram-. 

Ammonia  (U.  S.  P.,  sp.  gr.  0.9) 350.0      c.c. 

Glycerol     38.O      c.c. 

Distilled  water,  to  make  total  volume  1  liter. 

Roberts'  Reagent.0 — Mix  I  volume  of  concentrated  nitric  acid 
and  5  volumes  of  a  saturated  solution  of  magnesium  sulphate. 

Rosenheim's  Iodo-Potassium  Iodide  Solution.10 —Dissolve  2 
grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  ol 
.water. 

Salted  Plasma.11 — Allow  arterial  blood  to  run  into  an  equal  vol- 

I  Obermayer's  test,  page  281. 
-Experiments  on  blood  plasma,   page  201. 

3  Herter's  para-dimethylaminobenzaldehyde  reaction,  page  17° 

'Detection  of  hydrogen  peroxide,  page  226. 

"Topfer's  method,  page  413. 

c  Phenylhydrazine  reaction,  pages  24  and  306. 

7  Phenylhydrazine  reaction,  pages  24  and  306. 

s  Purdy's  method,  page  .170. 

"Roberts'  ring  test,  pages  97  and  315. 

"Rosenheim's  periodide  test,  page  252. 

II  Experiments  on   Mood  plasma,   page  201. 


422  PHYSIOLOGICAL    CHEMISTRY. 

ume  of  a  saturated  solution  of  sodium  sulphate  or  a  10  per  cent 
solution  of  sodium  chloride.  Keep  the  mixture  in  the  cold  room 
for  about  24  hours. 

Schiff's  Reagent.1 — This  reagent  consists  of  a  mixture  of  three 
volumes  of  concentrated  sulphuric  acid  and  one  volume  of  10  per 
cent  ferric  chloride. 

Schweitzer's  Reagent.2 — Add  potassium  hydroxide  to  a  solution 
of  cupric  sulphate  which  contains  some  ammonium  chloride.  Fil- 
ter off  the  precipitate  of  cupric  hydroxide,  wash  it  and  bring  3 
grams  of  the  moist  cupric  hydroxide  into  solution  in  a  liter  of  20 
per  cent  ammonium  hydroxide. 

Seliwanoff's  Reagent.3 — Dissolve  0.05  gram  of  resorcin  in  100 
c.c.  of  dilute  (1:2)  hydrochloric  acid. 

Sherrington's  Solution.4 — This  solution  possesses  the  follow- 
ing formula : 

Methylene-blue    0.1  gram. 

Sodium  chloride  1.2  gram. 

Neutral  potassium  oxalate   1.2  gram. 

Distilled  water    300.0  grams. 

Sodium  Acetate  Solution.5 — Dissolve  100  grams  of  sodium 
acetate  in  800  c.c.  of  distilled  wtaer,  add  100  c.c.  of  30  per  cent 
acetic  acid  to  the  solution  and  make  the  volume  of  the  mixture  up 
to  1  liter  with  distilled  water. 

Sodium  Alizarin  Sulphonate.0 — Dissolve  1  gram  of  sodium  aliz- 
arin sulphonate  in  100  c.c.  of  water. 

Sodium  Sulphide  Solution.7 — Saturate  a  one  per  cent  solution 
of  sodium  hydroxide  with  hydrogen  sulphide  gas  and  add  an  equal 
volume  of  one  per  cent  sodium  hydroxide. 

Solera's  Test  Paper.s — Saturate  a  good  quality  of  filter  paper 
with  0.5  per  cent  starch  paste  to  which  has  been  added  sufficient 
iodic  acid  to  make  a  1  per  cent  solution  of  iodic  acid  and  allow  the 
paper  to  dry  in  the  air.  Cut  it  in  strips  of  suitable  size  and  pre- 
serve for  use. 

1  Schiff's  reaction,  pages  159  and  252. 

2  Schweitzer's   solubility  test,  page  50. 

3  Seliwanoff's  reaction,  pages  35  and  339. 

4  "  Blood  counting,"  page  212. 

5  Uranium  acetate  method,  page  389. 
6T6pfer's  method,  page  413. 

7  Kriiger  and  Schmidt's  method,  pages  374  and  406. 
s  Solera's  reaction,  page  57. 


APPENDIX. 

Spiegler's  Reagent.1    -This  reagent  lias  the  following  comp 

tion : 

Tartaric   acid    

Mercuric    chloride    rams. 

Glycerol    100  g 

Distilled  water  to 

Standard  Ammonium  Thiocyanate  Solution.-'  This  solu- 
tion  is  made  of  such  a  strength  that  i  c.c.  of  it  is  equal  to  i  c< 
the  standard  argentic  nitrate  solution  mentioned  below.  To  pre 
pare  the  solution  dissolve  12.9  grams  of  ammonium  thiocyanate, 
NH4SCN.  in  a  little  less  than  a  liter  of  water.  In  a  small  lla^k 
place  20  c.c.  of  the  standard  argentic  nitrate  solution,  5  c.c.  of  a 
cold  saturated  solution  of  ferric  alum  and  4  c.c.  of  nitric  acid  (sp. 
gr.  1.2),  add  water  to  make  the  total  volume  [00  c.c.  and  thor- 
oughly mix  the  contents  of  the  flask.  Mow  run  in  the  ammonium 
thiocyanate  solution  from  a  burette  until  a  permanent  bi 
tinge  is  produced.  This  is  the  end-reaction  and  indicates  that  the 
last  trace  of  argentic  nitrate  has  been  precipitated.  Take  the  burette 
reading  and  calculate  the  amount  of  water  necessary  t<>  use  in  dilut- 
ing the  ammonium  thiocyanate  in  order  that  10  c.c.  of  this  solu- 
tion may  be  exactly  equal  to  10  c.c.  of  the  argentic  nitrate  solution. 
Make  the  dilution  and  titrate  again  to  be  certain  that  the  solution 
is  of  the  proper  strength. 

Standard  Argentic  Nitrate  Solution.3 — Dissolve  29.06  grams  of 
argentic  nitrate  in  1  liter  of  distilled  water.  Each  cubic  centimeter 
of  this  solution  is  equivalent  to  0.01  gram  of  sodium  chloride  or 
to  0.006  gram  of  chlorine. 

Standard  Uranium  Acetate  Solution.4 — Dissolve  35.461  grams 
of  uranium  acetate  in  I  liter  of  water.  One  c.c.  of  such  a  solution 
should  be  equivalent  to  0.005  gram  of  P2<  >5,  phosphoric  anhydride. 

This  solution  may  be  standardized  as  follows:  To  50  c.c.  oi  a 
standard  solution  of  disodium  hydrogen  phosphate,  of  Mich  a 
strength  that  the  50  c.c.  contains  o.  t  gram  of  P2<  >5,  add  5  C.C.  of  the 
sodium  acetate  solution  mentioned  on  p.  422  and  titrate  with  the 
uranium   solution    to   the   correct   end-reaction    as    indicated    in    the 

'Spiegler's  ring  test,  pages  98  and  316. 

"Volhard-Arnold  method,  page  396,  and  Clark's  modification  of  Dehn's 
method,  page  394. 

8  Volhard-Arnold  method,  page  396.  Mohr's  method,  page  39S1  •in,i  Clark's 
modification  of  Dehn's  method,  page  394. 

'Uranium  acetate  method,  page  389- 


424  PHYSIOLOGICAL    CHEMISTRY. 

method  proper  on  p.  389.  Inasmuch  as  1  c.c.  of  the  uranium  solu- 
tion should  precipitate  0.005  gram  °f  P2O5,  exactly  20  c.c.  of  the 
uranium  solution  should  be  required  to  precipitate  the  50  c.c.  of  the 
standard  phosphate  solution.  If  the  two  solutions  do  not  bear  this 
relation  to  each  other  they  must  be  brought  into  the  proper  relation 
by  diluting  the  uranium  solution  with  distilled  water  or  by  increas- 
ing its  strength. 

Starch  Iodide  Solution.1 — Mix  0.1  gram  of  starch  powder  with 
cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into 
75-100  c.c.  of  boiling  water,  stirring  continuously.  Cool  the  starch 
paste,  add  20-25  grams  of  potassium  iodide  and  dilute  the  mixture 
to  250  c.c.  This  solution  deteriorates  upon  standing,  and  therefore 
must  be  freshly  prepared  as  needed. 

Starch  Paste.2 — Grind  2  grams  of  starch  powder  in  a  mortar 
with  a  small  amount  of  water.  Bring  200  c.c.  of  water  to  the  boil- 
ing-point and  add  the  starch  mixture  from  the  mortar  with  con- 
tinuous stirring.  Bring  again  to  the  boiling-point  and  allow  it  to 
cool.  This  makes  an  approximate  1  per  cent  starch  paste  which 
is  a  very  satisfactory  strength  for  general  use. 

Stokes'  Reagent.3 — A  solution  containing  2  per  cent  ferrous 
sulphate  and  3  per  cent  tartaric  acid.  When  needed  for  use  a  small 
amount  should  be  placed  in  a  test-tube  and  ammonium  hydroxide 
added  until  the  precipitate  which  forms  on  the  first  addition  of  the 
hydroxide  has  entirely  dissolved.  This  produces  ammonium  fer- 
rotartrate  which  is  a  reducing  agent. 

Suspension  of  Manganese  Dioxide.4 — Made  by  heating  a  0.5 
per  cent  solution  of  potassium  permanganate  with  a  little  alcohol 
until  it  is  decolorized. 

Tanret's  Reagent.5 — Dissolve  1.35  grams  of  mercuric  chloride 
in  25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium 
iodide  dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up 
to  60  c.c.  with  distilled  water  and  add  20  c.c.  of  glacial  acetic  acid 
to  the  mixture. 

Tincture  of  Iodine.6 — Dissolve  70  grams  of  iodine  and  50  grams 
of  potassium  iodide  in  1  liter  of  95  per  cent  alcohol. 

Toison's  Solution.7 — This  solution  has  the  following  formula : 

1  Fehling's  method,  page  367. 

2  Experiments  on  starch,  page  44. 

3  Haemoglobin,  page  203.      Hsemochromogen,  page  202. 
*  Kriiger  and  Schmidt's  method,  pages  374  and  406. 

5  Tanret's  test,  pages  98  and  317. 

6  Smith's  test,  pages   155  and  325. 
'  "  Blood  counting,"  page  212. 


APPENDIX. 

Methyl  violet  0.025  gram. 

Sodium  chloride  •  <,  gram. 

Sodium  sulphate    ams. 

Glycerol  30.0  g  1 

Distilled   water    1O0.0  gi 

Topfer's  Reagent.1  I  )issolve  0.5  gram  of  di-methylamino- 
azobenzene  in  too  c.c.  of  95  per  cent  alcohol. 

Tropaeolin  OO.2 — Dissolve  0.05  gram  of  tropaeolin  <  )< )  in  [00 
c.c.  of  50  per  cent  alcohol. 

Uffelmann's  Reagent.'1 — Add  a  5  per  cent  solution  of  ferric 
chloride  to  a"i  per  cent  solntion  of  carbolic  acid  until  an  amethyst- 
blue  color  is  obtained. 

1  Topfer's  method,  page  413. 

2  Test  for  free  acid,  page  124. 

3  Uffelman's  reaction,  page  129. 


NDEX. 


Acacia    solution,    formation    of    emulsion 

by,  133 
Acetone,   305,   327 

formula  for,  327 

Gunning's  iodoform  test  for,  328 

Legal's   test   for,    329 

Lieben's  test  for,  329 

quantitative  determination  of,  400 

Reynolds-Gunning  test  for,  329 

Taylor's  test  for,  329 
Acholic  stool,  173 
Achroo-dextrins,    44,    55.    58 

a-achroo-dextrin,  55 

/3-achroo-dextrin,  55 

7-achroo-dextrin,    55 
Acid,  acetic,  265,  291 

alloxyproteic,  264,  285,  342 

amino-acetic,  67 

amino-ethyl-sulphonic,    151,   240 

a-amino-/3-hydroxy-propionic,   69 

a-amino-/3-imidazol-propionic.   74 

a-amino-iso-butyl-acetic,    76 

a-amino-methyl-ethyl-propionic,    77 

a-ainino-normal   glutaric,   79 

a-amino-propionic,  68 

amino-succinic,  78 

a-amino-iso-valerianic,    75    (see 
Valine) 

aspartic,   63 

benzoic,  161,  264,  289 

butyric,   119,   225,   265,  290 

caproic.   218 

carbamic,   184 

cholic,    151 

chondroitin-sulphuric,  232,  264,  285 

citric,  218 

combined   hydrochloric.    59 

cyanuric,   267 

a-e-di-amino-caproic,   77 

a-amino-/3-thiolactic,  disulphide  of,  72 

diaminotrihydroxydodecanoic,  63,  81 

diazo-benzene-sulphonic,  342 

ethereal  sulphuric,   162,  264,  279 

fatty,  131,  132,  138 

formic,  265,  291 

free  hydrochloric,   119,   123 

glutamic,   63 

glycocholie,    151 

glycuronic,  37 

glycerophosphoric,  248,  249,  265,  291 

glyoxylic,  91 

guanidine-a-ami  no- valerianic.    75 

hippuric,   160,   161,  264,  281,  383 

homogentisic,  28,  264,  2S8 

indole-amino-propionic.  73 

indoxyl-sulphuric,    162,   279 

inosinic,   236,   241 

kvnurenic,  264,  288 


Acid,   lactic.    [19,    1 29,    236 
lauric,  218 
mucic,    •.'..    !•..  .137 
myristic,  218 
nucleic,   107 
osraic,  251,  281 
oxalic.   264,  284 
oxaluric,   264,   290 
oxymandelic,  264,  288 
oxyproteic,   204,  jX- 
palmitic, 

para-cresol-sulphuric.   264,  279 
para-oxyphenyl-acetic,    162,    168,  264, 

287 
para-oxyphenyl-a-amino-propionic,  70 
para-oxyphenyl-propionie.      7..,      162, 

168,  264,  287 
paralactic,  237,  265,  291 
phenaceturic,  265.  291,  383 
phenol-sulphuric,  264,  279 
phenyl-a-amino-propionic,   69 
phosphocarnic,  236.  241.  265,  292 
phosphoric,  299 

pyrocatechin-sulphuric.    264,   279 
a-pyrrolidine-carboxylic,  80 
sarcolactic,  237 
skatole  acetic,  73 
skatole-carbonic,   167 
skatoxyl-sulphuric.  264.  279 
sulphanilic,   342 
tannic,   46,  49 
taurocholic,    151 
uric,  28,  236,  264 
uroferric.  264,  285.  342 
uroleucic.   264,  288 
volatile  fatty,  162,  165.  265,  290 
Acid  albuminate.     See  Acid  metaprotein. 
Acid  infraprotein.     See  Acid  metaprotein. 
Acid  metaprotein.    no 

coagulation  of,   110.   111 
experiments  on.    1  1  <■ 
precipitation  of,   1  1  1 
preparatii m  of,   no 
solubility  of.    1 10 
sulphur  content   of.    1  10 
Acidity  of  gastric  juice,  quantitativi 
termination  of.  413 
urine,  cause  of,   256,   .;■>" 

quantitative     determination     of, 
404 
Acidosis,  cause  of,  332 
\cid  li.emat in.   206 
\ercc- Rosenheim    formaldehyde   reaction. 

Acrolein,    formation    of,    from    olive    oil. 

135 
from    yjy. 
Activation.  5,   [42 


427 


428 


INDEX. 


Activation  by  calcium  salts,   142 
Adams'  paper  coil  method  for  determina- 
tion of  fat  in  milk,  410 
Adamkiewicz    reaction,    91 
Adenine,  241,  265 
Adipocere,  134 
Adler's  benzidine  reaction  for  blood,  192, 

196,   323 
Agglutination,  196 
Alanine    63,  68 
Albumin,  egg,  101 

powdered,  preparation  of,   101 

tests  on,  101 
serum,  86,  89,    182,   305,   313 
Albumin  in  urine,  305,  314 

acetic    acid    and    potassium    fer- 

rocyanide  test  for,  317 
coagulation    or    boiling   test    for, 

316 
Heller's  ring  test   for,   314 
Jolles'  reaction  for,  316 
Roberts'  ring  test  for,  315 
sodium   chloride  and  acetic  acid 

test  for,   317 
Spiegler's  ring  test  for,  316 
Tanret's  test  for,  317 
tests  for,  314 
Albumins,  86,  88,  89 
Albuminates.     See  Metaproteins. 
Albuminates,    formation    of,    by    metallic 

salts,   95,   97 
Albuminoids,   86 
Albumoids,   106 
Albumoscope,  97,  315 
Albumoses   (see  Proteoses,  p.   113) 
Alcohol-soluble  proteins.      See   Prolamins 
Aldehyde,  21,  26 
Aldehyde  group,  39 
Aldehyde  test  for  alcohol,  43 
v.   Aldor's   method   of   detecting   proteose 

in  urine,  320 
Aldose,  21 
Alkali     albuminate.        See     Alkali     meta- 

protein. 
Alkali-haematin,  206 
Alkali  metaprotein,  no,  in 
experiments  on,   in 
precipitation  of,   111 
preparation  of,   in 
sulphur  content  of,  no 
Allantoin,  264,  285 

crystalline   form  of,   286 
experiments  on,  286 
formula  for,  285 

preparation    of,   from   uric   acid,    286 
quantitative  determination  of,  407 
separation   of,   from  urine,   286 
Allen's  modification  of  Fehling's  test,  311 
Almen's  reagent,  preparation  of,  321 
Alloxyproteic  acid,  264,  285,  342 
Aloin-turpentine  test  for  "  occult  blood," 

175,  178 
Amandin,  86 
Amide   nitrogen,    62 

Amidulin.       See    Soluble   starch,    8,    54 
Amino  acids,  63,   162 


Amino  group,   88,   92 
a-amino-/3-hydroxypropionic   acid,   69 
a-amino-j8-imidazol-propionic   acid,   74 
a-amino-iso-butyl-acetic  acid,  76 
ct-amino-normal-glutaric  acid,   79 
Amino-succinic  acid,   78 
a-amino-iso-valerianic   acid,    75 
Ammonia,  67,   105 
Ammonia  in  urine,  257,  265,  295 

quantitative     determination     of, 
380 
Ammoniacal    silver    solution,    preparation 

of,  407 
Ammoniacal-zinc    chloride    test    for    uro- 
bilin, 293 
Ammonium  magnesium  phosphate 

("Triple  phosphate  "),  247 
301 
in  urinary  sediments,  344 
Ammonium  urate,  271,   347,   372 

crystalline    form    of,    Plate    VI, 
opposite  p.  348 
Amphopeptone,  113 
Amylase,  pancreatic,   141 

digestion  of  dry  starch  by,    143,    148 

inulin  by,   148 
experiments  on,  8,   147 
influence  of  bile  upon  action  of,  148 
metallic  salts,  upon  action  of,  147 
most    favorable    temperature    for    ac- 
tion of,   147 
salivary,   54,    119 

activity  of,  in  stomach,  55,   119 
experiments  on,   8 
inhibition  of  activity  of,  55 
nature  of  action  of,  54 
products  of  action  of,  55 
vegetable,    9 
Amylases,   3 

experiments  on,  8 
Amyloid,  49 
Amylolytic  enzymes.     See  Amylases 

quantitative  determination  of  ac- 
tivity of,  16 
Animal  parasites  in  feces,   175,   177 

in  urinary  sediments,  351,  361 
Anti-albumid,    122 
Anti-enzymes,  7 

experiments  on,  15 
Anti-pepsin,  8,    1 5 
Antipeptone,    113 
Anti-rennin,   7 
Anti-trypsin,  8,  15 
Appendix,  416 
Arabinose,  21,  37 

orcin  test  on,  38 
phenylhydrazine  test  on,   38 
Tollens'   reaction   on,    37 
Arginine,  63,   75,   141 

Arnold-Lipliawsky    reaction    for    diacetic 
acid,  331 
reagent,  preparation  of,  331 
Aromatic  oxyacids,   264,   287 
Ascaris,   15,   16 
Asparagine,    79 
Aspartic  acid,   63,   78,   80,   141 


[NDEX. 


429 


Aspartic  acid,  crystalline   form  of,  78 

formula   for,  78 
Ash  of  milk,  quantitative  determination 

of,  41 1 
Assimilation   limit,   23 

Atkinson    and    Kendall's   lia-niin    teat,    197 
Autolytic   enzymes,   3 

Bardach's  reaction,  94 

Barfoed's  reagent,  preparation  of,   12,  31, 

313 
Barfoed's    test    for    monosaccharides,    31, 

313 
Baryta    mixture,   preparation    of,   269 
Bayberry  tallow,   saponification  of,   136 

source  of,   136 
Bayberry  wax.     See  Bayberry  tallow,  136 
Beckmann-Heidenhain    apparatus,   260 
"  Bence  Jones'  protein,"  detection  of,  320 
Benedict's    method    for    quantitative    de- 
termination of  sugar,  368 
Benedict's  modifications  of  Fehling's  test, 
27,  28 
solutions,  preparation  of,  27,  28 
solution,   for  use  in   quantitative   de- 
termination   of    sugar,    preparation 
of,  368 
Benedict    and    Gephart's    method    for   the 
quantitative  determination  of  urea,  379 
Benzidine    reaction,    Adler's,    for    blood, 

192,    196,   323 
Benzoic  acid,  161,  269,  289 

crystalline   form   of,   289 
experiments   upon,    289 
formula   for,  289 
solubility  of,  289 
sublimation  of,  290 
Berthelot-Atwater  bomb  calorimeter,   388 
Bergell's    method    for    determination    of 

/3-oxybutyric  acid,  404 
Bile,   150,  305,  324 

constituents  of.   151 

daily  secretion  of,   151 

freezing-point   of.    151 

influence  on  digestion,  gastric,  128 

pancreatic.   146,    148 
inorganic  constituents  of,   151,   154 
nucleoprotein    of,    154 
reaction  of,  150,  154 
secretion   of,   150 
specific  gravity  of,   151 
Bile  acids,   151 

Guerin's  reaction  for,  156 
Hay's  test  for,  157 
Mylius's  test  for.   156 
Xeukomm's  test  for,   156 
Pettenkofer's  test  for.   156 
tests   for,   156 

v.  Udransky's  test  for.   156 
Bile  acids  in  feces,  detection  of,   179 
Bile  acids  in   urine.   325 

Hay's  test  for.  326 
Mylius's  test   for,  326 
Neukomm's    test   for,    326 
Pettenkofer's   test   for.    325 
tests  for,  325 


Bile   a.  ids    in    urine,    v.    Udran 

l-.r. 

Bile  pigments,   1 

<  rmelin'fl  '•    I  foi 
Harm 

I  luppcrt's    1.  a.  ip. n    for, 

1  55 
Smith's    test    for,    155 
tesl   for.    154,   '55 
Bile  pigments  in  urini 
Gmi 
llainiii.-n  :.,r. 

325 
I  [uppert* 

Xakayama's      reaction       lor. 
3-'4 

Rosenbach's  tesl   for 

Salkowski's  tesl    for 
Salkowski-Schippcr's      i 

tion  for,  325 
Smith's  test  for,  324 
tests  for,  324 
Bile  salts.  6,   151 

crystallization   of,    [52,    i?r 
Biliary,   calculi,    154 

analysis  of,    158 
Bilicyanin,    152,    154 
Bilifuscin,   152 
Bilihumin,    152 
Biliprasin,    152 
Bilirubin,   152,   153 

crystalline  form  of,   153 
in   urinary  sediments,  350 
Biliverdin,  152,   154 
"  Biological  "  blood  test,  193 
Bismuth  test  for  choline,  253 
Biuret,  92,  267 

formation  of,   from   urea.   92,   269 
Biuret    potassium    cupric    hydroxid.      See 
Cupri-potassium  biuret,  92 
test,  92 

Posner's  modification  of,  93 
Black's     method     for    determination    of 
/3-oxybutyric  acid,  403 
reaction  for  /3-oxybutyric  acid.  332 
reagent,  preparation  of,  333 
Blood,    i8j.  321 

agglutination  of.    [96 
Bordet   test   for,   193 
clinical  examination   of.  207 
coagulation   of,    191 
constituents  of,    182,    184 
defibrinated.   192 
detection  of.  192,  20. 
erythrocytes   of.    1  s 4 

experiments   oil,    193 

form  elements  of.  1 82 
guaiac  test  for,  178,  192,  196 
h.cmin   test    for.    [92,    197 
oxyhemoglobin   of,   185 

"occult,"    in    feces.    175.    178 
in    urine.    305,    .;.'  1 

leuci  190 

medico-legal  tests  for.   102 
microscopical    examination    01 
202 


430 


INDEX. 


Blood,   nucleoprotein   of,    182 

pigment  of,   185 

plaques,  182,  191 

plasma,  184 

preparation  of  hsematin  from,  199 

preparation  of  laky,  195 

quantitative   analysis   of,   415 

reaction  of,  182,  193 

serum,   184,  200 

specific  gravity  of,   182,   193 

spectroscopic   examination   of,    203 

test  for  iron  in,  195 

total  amount  of,  182 

v.    Zeynek   and   Nencki's    hsemin    test 
for,   199 
Blood  casts  in  urine,  351,  358 
Blood  corpuscles,   182,    184 

"  counting,"   212 
Blood  dust,  182,  191 
Blood  in  urine,  305,  321 

Adler's    benzidine    reaction    for, 

323 
guaiac  test  for,  322 
Teichmann's  hsemin  test  for,  322 
Heller's  test  for,  321 
Heller-Teichmann    reaction    for, 

322 
Schalfijew's  haemin  test  for,  322 
Schumm's  modification  of  guaiac 

test   for,    323 
spectroscopic  examination  of,  323 
tests  for,   321 

v.    Zeynek    and    Nencki's    hasmin 
test  for,  322 
Blood  plasma,   182,  201 

constituents  of,  182 
crystallization  of  oxyhemoglobin 

of,  189,  201 
effect    of    calcium    on    oxalated, 

201 
experiments  on,  201 
preparation    of   fibrinogen    from, 
201 
oxalated,  201 
salted,  201 
Blood  serum,   184,  200 

coagulation  temperature   of,   200 
constituents   of,    184 
experiments   on,    200 
precipitation  of  proteins  of,  200 
separation       of       albumin       and 

globulin  of,  201 
sodium  chloride  in,  201 
sugar  in,  201 
Blood   stains,   examination   of,   202 
Boas'  reagent,  as  indicator,   124  , 

preparation  of,   124 
Boekelman  and   Bouma's  method  for  de- 
termination   of   /3-oxybutyric    acid,   404 
Boettger's  test  for  sugar,   30 
Bomb  calorimeter,   Berthelot-Atwater,  388 
Bone,   constituents  of,   233 

ossein  of,  preparation  of,  233 
Bone  ash,  scheme  for  analysis  of,  234 
Borchardt's  reaction  for  lsevulose,  35,  338 


Bordet  test,  detection  of  human  blood  by, 

193 
Boric  acid  and  borates  in  milk,  detection 

of,   22(1 
Buccal  glands,  53 
Buffy  coat,   formation  of,    184 
Bunge's  mass  action  theory,   119 
Butyric  acid,   119,  225 
Butyrin,    132 
Bynin,   86,   105 

Cadaverin,  77 

Calcium    and    magnesium    in    urine,    265, 
303 
carbonate  in  urinary  sediments,  344 

345 
casein,   121 
oxalate,  344 

in  urinary  sediments,  344 
phosphate  in  urinary  sediments,  344 

in   milk,   224 
sulphate    in    urinary    sediments,    344 

346 
Calculi,  biliary,   154,   158 
urinary,   362 

calcium  carbonate  in,  363 

oxalate  in,  363 
cholesterol  in,  365 
cystine  in,   363 
fibrin  in,   363 
indigo   in,   365 
phosphates  in,  363 
uric  acid  and  urates  in,  363 
urostealiths   in,   363 
xanthine    in,    363 
Calliphora,    larvae    of,    formation    of    fat 

from  protein  by,   134 
Cane  sugar  (see  sucrose,  p.  41) 
Caproic  acid,   218 
Carbamic  acid,   184 
Carbohydrates,  21 

classification  of,  21 
composition  of,  21,  22 
review  of,  50 

scheme  for  detection  of,   51 
variation  in  solubility  of,  22 
Carbonates  in  urine,  265,   303 
Carbon  moiety  of  protein  molecule,  134 
Carbon  monoxide,  haemoglobin,  205 

tannin  test  for,  205 
Carboxyl   group,   88 
Carnine,    236 
Carnitine,  236 

formula  for,   240 
Carnomuscarine,   236 
Carnosine,  236,  240 
Cartilage,   232 

constituents   of,   232 
experiments  on,  232 
Hopkins-Cole  reaction  on,  232 
loosely  combined  sulphur  in,  232 
Millon's  reaction  on,   232 
preparation   of  gelatin   from,   232 
solubility   of,   232 
xanthoproteic  test  on,   232 
Casein,  219 


INDEX. 


Casein,  soluble,   2  i  9 

calcium,    121 

quantitative  determination   of,  412 
Caseinogen,  87,  88,  107,  180,  21m 

action  of  rennin  upon,    ui.   2ig 

biuret  test  on,  224 

Millon's   test   on,   224 

precipitation  of,  223 

preparation  of,  223 

solubility  of,  224 

test  for  loosely  combined  sulphur  in, 
224 

test  for  phosphorus  in,  224 
Casts,  351,  354 

blood,    351,    356,    358 

epithelial,  351,  358 

fatty,   351,   358 

granular,  351,  356 

hyaline,  351,  355 

pus,  351,  359 

waxy,  351,  358 
Casts   in    urinary   sediments,   351,    354 
Cat  gut,   127 
Catalase,    14 

experiments  on,  14 
Catalysis,  2 
Cellulose,  22,  49 

action    of    Schweitzer's    reagent    on, 
49 

hydrolysis    of,   49 

iodine  test  on,  49 

solubility  of,  49 
Cellulose  group,  22 
Cerebrin,  248,  250 

experiments  on,  252 

hydrolysis  of,  252 

microscopical  examination   of,   252 

preparation   of,   252 

solubility  of,  252 
Cerebro-spinal  fluid,  choline  in,  249 
Charcot-Leyden    crystals,    175 

form   of,    174 
Chlorides  in  urine,  265,  298 
detection  of,  299 
quantitative     determination     of, 

394 
Cholecyanin,   154 
Choleprasin,    152 

Cholera-red  reaction   for  indole,    169 
Cholesterol,    154,    158 

crystalline  form  of,   159 

formula   for,   250 

iodine-sulphuric    acid    test    for,    158, 

251 
isolation  of,  from  biliary  calculi,   158 
Liebermann-Burchard    test    for,    158, 

251 
occurrence  of,  in  urinary  sediments, 

344.  349 
preparation   of.   from   nervous   tissue, 

25 1 
Salkowski's  test  for.  150.  252 
Schiff's  reaction  for,  159,  252 
tests  for,   158,  251 

Choletelin,  152 

Choline,  248.  252 


1  holine,  formula   i"i 

for,  252 
( Ihondrigt  1 
( Ihondroalbumoid 

<  bondromucoid 
Chondroitin,  232 

Chondroitin  Blllphuril  „•,   264,   285 

1  hondrosii 

Chromoproteins,  85,  see  Haemoglobin* 

<  ipollina's   test,   25,   .0.7 

Clark's    modification    of    Dehn'a    method 

for  determination  of  chlorides, 
Cleavage    products    of    protein     (see    De- 

composition   products),  61 
Clupeine,  87,  88 
Coagulated  proteins,  88,  1 1 1 
biuret  test  on,   1  1  2 
formation  of,   1 1 1 
I  [opl  ins-Cole  reaction  on,  1  12 
Millon's  reaction  on,  '1 12 
solubility  of,   112 
xanthoproteic    reaction    on.     1:2 
Coagulation  of  proteins,  99,  111 

changes    in    composition   during, 

100,    112 
fractional,   100,   112 
Coagulation  temperature  of  proteins.  100, 
1 12 
apparatus    used    in    determining, 

100 
method    employed    in    determin- 
ing,   100 
Co-enzyme,    5 
Collagen,  86,   106,  232,  248 
experiments  on,  229 
percentage  of,  in  ligament.  231 

in   tendon,   228 
production    of   gelatin    from,    229 
solubility  of.  229 
transformation    of,   228 
Colostrum,   221 

microscopical    appearance    of.    219 
Combined  hydrochloric  acid.   119 

tests  for,  1 23- 1  25 
Compound  test  for  lactose  in  urine.  337 
Congealing-point   of   fat,    139 
Congo  red,  as  indicator,   124 
preparation  of,    124 
Conjugated  proteins,  87,  89,   106 
classes  of,  87,  89,  106 
experiments    on.    195,    196 

20 1.    203,   223 
nomenclature  of,  87 
occurrence  of,    106 
Conjugate  glycuronati 

fermentation  reduction    u  - 

334 
Tollens'   reaction   on,   334 

( 'onnective  tissui 
Cowie's  guaiac  test,   178 
Creatine,  184,  -." 

crystalline  form  of, 

formula  for, 

quantitative  determination 

separation  of,  from  meat  extr.: 
Creatinine,   28,  236, 


432 


INDEX. 


Creatinine,  coefficient,  definition  of.  276 

crystalline  form  of,  277 

daily    excretion    of,    276 

experiments  on,  277 

formula  for,  240,  275 

Jaffe's  reaction  for,  278 

quantitative   determination   of,  392 

Salkowski's  test  for,  278 

separation  of,  from  urine,  277 

Weyl's  test  for,  278 
Creatinine-zinc    chloride,     formation     of, 

276,  278 
Cresol,  para,   162 

tests  for,  170 
Cryoscopy,  259 
Cul-de-sac,   118 
Cupri-potassium  biuret,   formation  of,  92 

formula  for,  93 
Cyanuric  acid,   267 

formula  for,  267 
Cylindroids   in   urinary   sediments,    359 
Cystine,  63,  72,  141 

crystalline  form  of,  73 

detection  of,  349 

formula  for,   72 

in  urinary  sediments,  348 
Cytoglobulin,   87,   89 
Cytosine,    107 

Wheeler-Johnson  reaction  for,  107 

Dakin's   methods   for   quantitative   deter- 
mination of  hippuric  acid,  383 
Dare's  hsemoglobinometer,  210 
description  of,  210 
determination  of  haemoglobin  by, 
210 
Darmstadter's   method   for   determination 

of  (3-oxybutyric  acid,  403 
Deamidizing   enzyme,    3 
Decomposition    products    of    proteins,    61 
crystalline   forms   of,   68-81 
experiments  on,  82 
isolation  of,  82 
Degradation  products  of  protein  (see  De- 
composition products,  61 
Dehn's    method,    Clark's    modification    of, 

394 
Dehn's  reaction  for  hippuric  acid,  283 
Delusive  feeding  experiments,   118 
Derived  proteins,  87,  108 
Detection  of  preservatives  in  milk,  225 

boric  acid  and  borates,  226 

formaldehyde,   225 

hydrogen  peroxide,  226 

salicylic  acid  and  salicylates,  226 
Deuteroproteose,  88,  89 
Dextrin,   22,  48 

achroo-,  44,  55 

a-achroo-,  55 

/3-achroo-,  55 

7-achroo,  55 

erythro-,  44,  48,  55 

action  of  tannic  acid  on,  49 

diffusibility  of,  49 

Fehling's  test  on,  48 

hydrolysis  of,  48 


Dextrin,   iodine  test   on,   48 

solubility  of,  48 
Dextrosazone,   crystalline  form  of,   Plate 

III,  opposite  p.  24 
Dextrose,  21,  23,  305 

Allen's      modification      of     Fehling's 

test  for,  311 
Barfoed's  test  on,   31,   313 
Boettger's  test  on,  30,  311 
Cipollina's  test  on,   25,  307 
Benedict's  modifications  of  Fehling's 

test,   28,  29,   309,   310 
diffusibility  of,  25 
experiments  on,  23 
Fehling's  test  on,  27,  308 
fermentation  of,  313 
iodine  test  on,  25 
Molisch's   reaction  on,  23 
Moore's  test  on,  26 
Nylander's  test  on,   30,  312 
phenylhydrazine  test  on,  306 
quantitative  determination  of,   367 
reduction  tests  on,  26,  307 
solubility  of,  23 
Trommer's  test  on,  27,  308 
Dextrosazone,   crystalline  form   of,   Plate 

III,  opposite  p.  24 
Diacetic  acid,  305,  331 

Arnold-Lipliawsky   test   for,   331 
formula  for,  330 
Gerhardt's   test   for,    330 
quantitative     determination     of, 
401    ' 
Diamino  acid  nitrogen,  62 
Diaminotrihydroxydodecanoic    acid,    81 
a-e-di-amino-caproic  acid,   77 
Diastase   (see  Vegetable  amylase) 
Diazo-benzene-sulphonic   acid,   342 

reagent,  preparation  of,  342 
Diazo  reaction   (Ehrlich's),  342 
Differentiation   between   pepsin    and  pep- 

singen,    126 
Digestion,  gastric,  118 
pancreatic,  140 
salivary,   53 
Di-methyl-amino-azobenzene      (see     Top- 

fer's  reagent),   123 
Dipeptides,  64,  66,  88,  89 
Disaccharides,  38 

classification  of,  21 
Dissociation  products  of  protein  (see  De- 
composition products,  67) 
Doremus-Hinds  ureometer,  378 
Drying     method     for     determination     of 

total  solids  in  urine,  408 
Duodenum,  epithelial  cells  of,   140 

Earthy  phosphates  in  urine,  299,  302 

quantitative     determination     of, 
390 
Edestan,   18,  87,   109 

experiments  on,   109 
Edestin,  86,   103 

coagulation   of,    103 
crystalline  forms  of,   104 
microscopical  examination   of,    103 


[NDEX. 


Edestin,  Millon's  test  on,   103 

preparation  of,   103 

solubility  of,   103 

tests  on  crystallized,    [03 
filtrate  of,    104 
Ehrlich's     diazo-benzene-sulpbonic     acid 

reagent,  preparation  of,  342 
Ehrlich's  diazo  reaction. 
Ehrlich's    mechanical    eye-piece,    use    of, 

216 
Einhorn's  saccharometer,  31 
Elastin,  86,  88,   106,  231 

experiments  on,  231 

preparation    of,    231 

solubility  of,  231 
Electrical  conductivity  of  urine,  261 
Embryos,  glycogen  in,   237 
Enterokinase,  5,  141 
Enzymes,    1 

activation  of,   5 

adsorption   of,   4 

classification  of,  3 

definition    of,    1 

experiments  on,  8 

preparation    of,   4 

properties  of,  4 
Epiguanine,   265,   294 
Episarkine,  265,  294 

Epithelial  cells  in  urinary  sediments,  351, 
352 

casts  in  urinary  sediments,   351,   358 
Epithelial  tissue,   227 

experiments  on,  227 
Erepsin,    13 

experiments  on,   13 
Erythrocytes,   182,   184 

counting  the,  212 

diameter  of.  184 

form  of,   184 

influence  of  osmotic  pressure  on,  195 

in  urinary  sediments,  351,  360 

number  of,  per  cubic  mm.,   185 

of  different  species,  184 

stroma  of,    182,    185 

variation  in  number  of,   185 
Erythro-dextrin,  44,  55,  58 
Esbach's  albuminometer,  367 

method  for  determination  of  albumin, 
366 

reagent,  preparation  of,  366 
Ester,  definition  of,   131 

hydrochloric  acid,  of  haematin,  199 

sulphuric  acid,  of  haematin,  199 
Ethereal  sulphates,  296,  297 

quantitative     determination     of, 
386 
Ethereal  sulphuric  acid.   162,   264,  279 
Ethyl  butyrate  test  for  pancreatic  lipase, 

149 
Euglobulin,   182,   183 
Excelsin,  103 

crystalline  form  of,   105 
Extractives  of  muscular  tissue,   236 
nitrogenous,   236 
non-nitrogenous,  236 
29 


Fatigue  Bubstam  ea  "i  mu 

FatS,     1   ;i 

absorption  of,   133 

apparatus      \>>r      determination      --i 

melting  point  ol 
boiling  point  of,   133 
chemical  composition  of,  131 
congealing  point   "t 
crystallization  of,   133, 
digestion  of,  133 
emulsification   of, 
experiments  on,  135 
formation  <>f  from  protein 
formation  <>i  a<  rolein  from, 
hydrolysis  of, 
in  milk,  218,  jj., 
in  urine.   305,  ,\m 
melting  point  of,   1  ■; 
nomenclature  of,   133 
occurrence  of,   131 
permanent   emulsions  of,   133 
quantitative     determination     of,     in 

milk.    1  1  M 
rancid.    133 
reaction    of.    135 
saponification  of,  132,  136.   138 
solubility  of.   133.   135 
transitory  emulsions  of,   133 
Fat-splitting  enzymes  1  see  Lipas*  - 
Fatty  acid,   131,   132,   138 
Fatty    casts    in    urinary    sediments,    351, 

358 
Fatty   degeneration,    134 
Feces,  172 

blood  in,  175 
daily  excretion   of,    172 
detection  of  albumin  and  globulin  in. 
180 
bile   acids   in.    17') 
bilirubin    in.    179 
caseinogen    in,    180 
cholesterol    in.    177 
hydrobilirubin  in.    1  7<j 
inorganic   constituents   ol 
nucleoprotein   in,    180 
proteose  and  peptone  in. 
experiments  on,    176 
form  and  consistency  of,    174 
macroscopic  constituents  of,   174 
microscopic  constituents  of,   175 
odor  of,   1  73 
pigment  of,   173 
reaction  of,   1  74 

separation   .>t\   importance   of,    174 
Fecal    bacteria.    1  75 

Folding's    method    for    determination    of 
dextrose, 
Benedict's    modification 
of. 
solution,  preparation  ol 

test.    J 7.    308 

Allen's  modification  of,  31 1 
Benedict's    modificat  ii 

Ferments,  classification  •■!'.  1 
Fermentation  test.  31,  313 


434 


INDEX. 


Fermentation    method    for    determination 

of  dextrose,  371 
Fermentation-reduction      test      for      con- 
jugate glycuronates,  334 
Ferric    chloride    test    for    thiocyanate    in 

saliva,   57 
Fibrin,   183,   191,  202,  305 

in  urinary  sediments,  351,  361 
separation  of,  from  blood,  191,  202 
solubility  of,  202 
Fibrin  ferment,   184,   191 
Fibrinogen,   182,  191 
Fibroin,   silk,   66 
Fischer  apparatus,  67 

photograph  of,  71 
Fleischl's  hsemometer,  207,  208 
description  of,  207 
determination  of  haemoglobin  by, 
207 
Fleischl-Miescher  hsemometer,  209 
Fluorides  in  urine,  265,  298,  394 
Fly-maggots,  experiments  on,   134 
Folin-Hart  method   for  determination   of 
combined  acetone  and  diacetic 
acid,    397 
for     determination     of     diacetic 
acid,  401 
Folin-Messinger-Huppert  method  for  de- 
termination     of      diacetic 
acid,   401 
Folin's     method     for     determination     of 
acetone,  400 
acidity   of  urine,   404 
ammonia,  380 
creatinine,   392 
ethereal  sulphates,  386 
inorganic     sulphates,     385 
total  sulphates,  384 
urea,    377 
Folin-Shaffer    method    for    determination 

of  uric  acid,  372 
Foreign   substances   in   urinary   sediment, 

35i,  361 
Formation    of    methylphenyllsevulosazone, 

35 
Form  elements  of  blood,   184 
Formic  acid,  265,   291 
Fractional  coagulation  of  proteins,   112 
Free  hydrochloric  acid,   123 

tests  for,  123-125 
Freezing-point   of   bile,    151 

blood,   182 

milk,  218 

pancreatic  juice,   141 

urine,  260 
Fuchsin-frog  experiment,  242 
Fuld  and  Levison's  method  for  peptic  ac- 
tivity,  1 8 
Fundus  glands,   118 
Furfurol  solution,  preparation  of,   156 
Fusion  mixture,  preparation  of,    102 

Galactase,   221 

Galactose,  21,  36,  305,  337 

experiments  on,  36 
Gallic  acid  test  for  formaldehyde,  225 


Gastric  digestion,  118 

conditions  essential  for,  120,  125 
general  experiments  on,  125 
influence  of  bile  on,  128 
influence    of    different    tempera- 
tures on,  126 
most  favorable  acidity  for,  126 
power  of  different  acids  in,  127 
products  of,  120 
Gastric  fistula,  118 
Gastric  juice,   1 18-122 
acidity  of,    119 
artificial,  preparation  of,   122 
composition  of,  119 
enzymes   of,    119 
origin    of    hydrochloric    acid    of, 

119 
quantitative   analysis   of,   413 
quantity  of,   118 
reaction   of,    119 
specific  gravity   of,    119 
lactic  acid  in,  test  for,   129 
Gastric  lipase,   119,   121 
Gastric  protease,  1 
Gastric  rennin,   119,   121,  128 

action   of,   upon   caseinogen,    121 

219 
experiments  on,   128,    130 
influence    of,    upon    milk,    223 
in  gastric  juice,  absence  of,  121 
nature  of  action  of,   121 
occurrence  of,    121 
Gelatin,  91,  228,  230 
coagulation  of,  230 
experiments  on,  230 
formation  of,  229 
Hopkins-Cole  reaction  on,  230 
Millon's  reaction  on,  230 
precipitation  of,  by  alcohol,  230 
alkaloidal    reagents,    230 
metallic  salts,   230 
precipitation    of,    by    mineral    acids, 

230 
preparation  of,  from  cartilage,  232 

from  collagen,  229 
salting-out  of,  230 
solubility  of,  230 
Gerhardt's  test  for  diacetic  acid,  330 
Gerhardt's   test   for  urobilin,    293 
Gliadin,  86,   105 
Globin,   86,   88 
Globulins,  86,  99,  102 
experiments  on,   103 
preparation  of,  103 
serum,  86,  182,  305,  318 
in  urine,  305,  318 
tests  for,  318 
vegetable,   86 
Glucoproteins    (see   Glycoproteins,  p.    89) 
Glucose  (see  Dextrose,  p.  21) 
Glutamic  acid,  63,  79,  141 

formula  for,  79 
Glutelins,  86,  104 
Glutenin,  86,   104 
Glycerol,   132,   138 

borax  fusion  test  on,  139 


[NDEX. 


435 


Glycerol,  experiments  on,   138 

formula  for,   135 
Glycerol  extract  of  pig's  stomach,  prepar- 
ation of,   122 
Glycerophosphoric  acid,  248,  249,265,291 
Glycocholic  acid,    151 
Glycocholic  acid  group.  151 
Glycocoll,  63,  67,  t  5 f 

formula  for,  67,  151 

preparation  of,   160 
Glycocoll   ester  hydrochloride,   crystalline 

form  of,  68 
Glycogen,  22,  47,  236,  237 

experiments  on,   244 

hydrolysis  of,  244 

in    embryos,    237 

influence  of  saliva  on,  244 

iodine  test  on,  244 

preparation  of,  244 
Glycoproteins,  87,  106,  228 

experiments  on,  229 

hydrolysis  of,   229 
Glycosuria,   alimentary,   23 
Glycuronates,  conjugate,  28,  305,  309,  334 
Glycuronic  acid,  37 
Glycyl-glycine,  formation  of,  66 
Glyoxylic  acid,  91 

formula  for,  91 
Gmelin's  test  for  bile  pigments,  154,  324 
Rosenbach's  modification  of,  155, 

324  _ 
Granular  casts  in  urinary  sediments,  351, 

356 
Granulose.  43 

Green  stools,  cause  of,  173 
Gross'     method     for    quantitative     deter- 
mination of  tryptic  activity,   19 
Guaiac  solution,  preparation  of,   418 
Guaiac  test  on   blood,    178,    192.    196 
on  feces,    178 
milk,   222 
in  urine,  322 
Guaiac    test,    Schumm's    modification    of, 

196 
Guaiac  test  on  pus,  354 
Guanidine-a-amino-valerianic  acid,  75 
Guanidine-residue,  62 
Guanine,  236,  241 
Gums    and    vegetable    mucilage    group    of 

carbohydrates,   22 
Gunning's  iodoform  test  for  acetone,  328 
Giinzberg's  reagent,   as  indicator,    123 

preparation  of,  123 
Giirber's  reaction   for  indican,  281 

Hsematin,  108 

acid-,  206 

alkali-,  206 

preparation  of,   199 

reduced  alkali-,  206 
Hsematodir,  153.  173 

crystalline  form  of,   153.   173 

in  urinary  sediments.  344.  350 
Hematuria,  321 
Hsmatoporphyrin,   10.    190.  207.  305.   31a 

in  urine.   305,   336 


I  l.i'iinn   crystals,    form   of,    ioX 
"'7 

Haemochromogen,  108,   18s 

I  fa  1 \  .miii    8; 

1  '■'  "' ee   Blood  dust. 

1 1- globin    8;    89    106,  107 

■  11  bon  monoxide,  1  91 

di  composition  of,  185 

diffusion   of, 

met,   ton, 

nxy.    185,    igo,    - 

quantitative  determination  ol 
reduced,   203 
Haemoglobins,  87,  107 

I  la  1 1 1  ■ ' l'  1 . . 1 1  i  1 1 1 1  r i a ,   32  t 

Hammerschlag's    method    for   determina- 
tion of  specific  gravity  of  blood,   194 
Hammarsten's  reaction,  i$S,  325 

reagent,  preparation  of,  155,  325 
Hay's  test  for  bile  acids,   157, 
Heintz  method  for  determination  of  uric 

acid,  373 
Hclicoprotein.  87    • 
Heller's  test  for  blood  in  urine.  321 
Heller-Teichmann    reaction    for   blond    in 

urine,   322 
Heller's  ring  test   for  protein,  07.  314 
Hemi-cellulose,  22 
Htrter's     naphthaquinone     reaction      for 

indole,   168 
Herter's  para-dime  thy  lam  inobenzaldehyde 

reaction,  170 
Heteroproteose,  89 
Heteroxanthine,  265,  2^4 
Hexone  bases,  77 
Hexoses,  21,  22 

Hippuric  acid,   160.   161,  264.  281.  383 
crystalline  form  of,  282 
Dakin's    methods    for    quantita- 
tive determination  of.  383 
Dehn's  reaction   for. 
experiments   on,    160, 
formula  for,  161 
in    urinary    sediments,    54. j 
melting  point   1  if 
Roaf's  method  for  crystallization 

of,  283 
separation  of,  from  urin< 
solubility  of, 

I I  ippuric  acid,  sublimation  of, 

synthesis  of,   1  60 
Histidine,  63,  74.   '4' 

hydrochloride,  crystalline  form 
Knoop's  color  reaction   1   1 
rlistones,  86,  88 
I  [offmann's  reaction   for  tyn  • 
Homogentisic  acid.  28 
formula    for, 
1  [opkins  Cole  reaction,  <n 

on  solutions,  01 
on   solids.    101 
Hopkins  Cole  reagent,  prepara ti 
Hordein,  :<>.  St..  105 
Horismascope  -  -1 1    Vlbun    - 
Hormone,  definition  of, 


436 


INDEX. 


Hopkins'    thiophene    reaction    for    lactic 

acid,   129 
Hvifner's   urea   apparatus,   377 
Human  fat,  composition  of,  133 
Huppert's  reaction  for  bile  pigments,  155, 

324 
Hiirthle's  experiment,  247 
Hyaline  casts  in  urinary  sediments,  351, 

355 
Hydrobilirubin,  detection  of,  in  feces,  179 

extraction  of,   179 
Hydrochloric    acid    of    the    gastric    juice, 
119 
origin  of,  theories  as  to,   119 
Hydrochloric  acid  test  for  formaldehyde 

(Leach),  225 
Hydrogen  peroxide  in  urine,  265,   304 

detection  of,  in  milk,  226 
Hydrolysis  of  cellulose,  49 
cerebrin,    252 
dextrin,  48 
glycogen,  244 
inulin,  47 
proteins,  64 
starch,  46 
sucrose,  41 
Hyperacidity,    119 
Hypoacidity,   119 

Hypobromite  solution,  preparation  of,  375 
Hypoxanthine,  236,  245,  265,  294 

formula  for,  241 
Hypoxanthine    silver    nitrate,    crystalline 
form  of,  245 

Ichthulin,  87 
Ignotine,  236 

formula  for,  241 
Imide  bonds,  66 
Indican,  162,  279,  312 

formula  for,   163,  280 

Giirber's  reaction  for,  281 

Jaffe's  test  for,   280 

Lavelle's   reaction   for,    281 

Obermayer's  test  for,  281 

origin  of,    162,  279 

Rossi's  reaction  for,  281 
Indigo-blue,    163,  2.80 

formula  for,   163,  280 
Indigo  in  urinary  sediments,  344,  351 
Indole,    162 

formula  for,    162 

origin  of,   162 

test   for,    168 
Indole-amino-propionic  acid,  73 
Indoxyl,    162,   279,   280 

formula   for,    162,   280 
Indoxyl,  origin  of,   163,  279 

potassium  sulphate   (see  Indican,  pp. 
162-163,    279) 
Indoxyl-sulphuric  acid,  162,  279 

formula  for,  162,  280 
Infraproteins   (see  Metaproteins,  87) 
Inorganic    physiological    constituents     of 

urine,   295 
Inosinic  acid,  236 

formula   for,   241 


Inosite,  21,  305,   339 
formula  for,  339 
in  urine,  305,  339 
Intestinal  juice,    143 

enzymes   of,    143 
preparation  of,  143 
Inulase,  47 
Inulin,   22,   46 

action  of  amylolytic  enzymes  on,  47 

58. 
Fehling's  test  on,  47 

hydrolysis  of,  47 

iodine  test  on,  47 

reducing  power  of,  46 

solubility  of,  46,  47 

sources  of,  46 
Inversion,  41,  43 

Invertases,  experiments  on,   11,  144 
Invertin    (see   Sucrase,  p.   41) 
Inverting  enzymes,  3 
Invert   sugar,    41,    144 
Iodide  of  dextrin,  48 

of  starch,  44 
Iodine  test,   25,   44,   47,   48 
Iodine-sulphuric  acid  test  for  cholesterol, 

158,  251 
Iodoform  test  for  alcohol,  42 
Iodothymol   compound,    329 
Iron  in  blood,  189,  195 

detection  of,    195 

in  bone  ash,   234 

detection  of,  234 
Iron  in  protein,  62 
Iron  in  urine,  265,  303 

detection  of,  304 
Isoleucine,  77 
Isomaltose,  21,  40,  55 

Jaffe's  reaction  for  creatinine,  278 
Jaffe's  test  for  indican,  280 
v.  Jaksch-Pollak  reaction  for  melanin,  341 
Jejunum,  epithelial  cells  of,  140 
Jolles'  reaction  for  protein,  98,  316 

reagent,  preparation  of,  98,  316 
Juice,  gastric,   1 18-122 

pancreatic,   140-143 

intestinal,    143 

Kastle's  peroxidase  reaction,   222 
Kephalin,   248,   250 
Kephyr,  40 
Keratin,   86,   227 

experiments   on,   227 

solubility  of,  227 

sources   of,   227 

sulphur  content  of,  227 
Ketone,    21,   26 
Ketose,  21 
Kjeldahl    method    for    determination    of 

nitrogen,  381 
Knoop's   color   reaction   for   histidine,    74 
Knop-Hiifner     hypobromite     method     for 

determination   of  urea,   374,   376 
Konto's  reaction   for  indole,   169,   181 
Koppe's    electrolytic    dissociation    theory, 
119 


[NDEX. 


Koumyss,  40 

Kraut's  reagent,  preparation  of,  253 
Kriiger    and    Schmidt's    method    for    the 
quantitative    de 
termination       of 
purine  bases,    |"=, 

"i   uric  acid,  .?".? 
Kiilz's  test  for  /3-oxybutyric  acid,  ,$.u 
Kynurenic  acid,  264,  288 
formula   for,  288 
isolation   of,   from   urine,   289 
quantitative     determination     of, 
280 

Lactalbumin,  86,  218,   221 

quantitative  determination   of,    1 1 - 
Lactase,  12,  141,  143 

experiments  on,  12 
Lactic  acid,  40,  129,  236 

ferric  chloride  test  for,   129 
Hopkins'  thiophene  reaction  for, 

129 
in   muscular   tissue,   236,   237 
in  stomach  contents,    129,    130 
tests   for,    129 
Uffelmann's   test  for,    129 
Lacto-globulin,  218,  221 
Lactometer,      determination      of     specific 

gravity   of  milk   by,   410 
Lactosazone,    crystalline    form    of,    Plate 

III,  opposite  p.  24 
Lactoscope,   Feser's,   41 1 
Lactose,  21,  40 

experiments  on,  41 
fermentation  of,  40 
in   urine,   305,  336 
quantitative  determination   of,   412 
Lactosin   in  milk,   221 
Larvo-a-proline,   80 
Lsevulosazone,   crystalline   form   of,    Plate 

III,  opposite  p.  24 
Lsevulose,    21.    35 

Borchardt's  reaction  for,  35 
in  urine,  305,  338 

methyl-phenylhydrazine    test    for,    35 
Seliwanoff's  reaction  for,  35 
Laiose  in  urine,  305,  340 
Laked  blood,   182,   193 
Laky  blood,   195 
Laurie  acid,  218 
Laurin,   132 

Lavelle's  reaction  for  indican,  281 
Leach's  hydrochloric  acid  test   for   form 

aldehyde,    225 
Lecithans,  87 
Lecithin,   151,  248,  250 
acrolein  test  on,  251 
decomposition  of,  249 
experiments  on,  249 
formula  for,  249 

microscopical  examination  of,  250 
osmic  acid  test  on.  250 
preparation  of,  250 
test   for  phosphorus   in,   25J 
Lecithoproteins,  87.    ro8 
Legal's  reaction  for  indole,   169 


L 


L 


egal's  ievt    for  ... .  torn 

ell.    111.    ,     '.    :  .     ;       ,     76,     141 

tallint    form  >.i   impun . 

pun 
1  intents  on,  x.\ 
formula  for,  76 
in  urinary  sedii 

micros*  opi<  ..1  •  ■ nation  of 

eparation  of    fi 
solubility  of,  x.j 
sublimation  of,  84 
eucocytes,    1  82,    1  o.> 
counting  the,  215 
number  of,  per  1  •  1 J .  i «    mm., 
si/.-  of,   1  go 

variation   in   number  of,    190 
eucocytosis,    [90 
eucosin,  96 
eucyl  alanyl 

eucyl  leucine,  formation  of,  66 
ichenin,  22,  48 
ieben's  tesl    for  acetom 
ieberkuhn's     jelly     >  see      Vlkali     meta- 
proti  m.   p.    mm 

iehermann  Burchard     test     for     choles- 
terol, 158,  251 
iebermann's   reaction,   93 
ipase,  gastric,   ui 
ipase,   pancreatic.    10,    132 
experiments  on,    i" 
ethyl-butyrate  tesl  for,   149 
litmus-milk  tesl   for,   1  (8 
1  pases,  3,   10 

experiments  on,   10 
ipoids  of  nervous  tissue.   J4X.   250 
ipolytic   enzymes    (see   Lipases,   !• 

Litmus  milk  "   test    for  panereatic   lipase, 
.48 
ilgol's   solution,   preparation    .>t.   04 
ysine,  63,  ~7-  '41 
ysine    picrate,    crystalline    form    of,    78 


Magnesia  mixture,  preparation  ol 

Magnesium    in   urine.   265,   303 

phosphate  in  urinary  sediments 
350 
Maltase,   13  39,  55.   '44 
experiments   on.    13 
Maltosazone,    crystalline    form    "t.    Plate 

111.   opposite  p.   24 
Malti.se.    _'i.    30 

experiments  on,    p. 
structure  of,  30 
Marshall's  una  apparatus,  373 

Melanin   in   urine.   305, 

urinary  -e<lim<  nts,   3 1  s.  351 
Melting  poinl    apparatus.    1 38 

of  fats,  determination  of,  130 
Messinger-Huppert,    method     for 
mination     of    combined     acetone    ami 
diacetic  acid,  300 
Metaproteins,  87,   108,   109 
acid,  87 
alkali.    87 

experiments   on,    1 10 
precipitation  of,  no 


43« 


INDEX. 


Metaproteins,  sulphur  content  of,  no 
Methjemoglobin,   190,  206 
Methylene  blue,   127 
Methyl-mercaptan,  162,  173 
Methyl-pentose    (see   Rhamnose,   p.   21) 
Methylphenylhydrazine,  36 
Methylphenyllaevulosazone,    formation   of, 

35 
i-methylxanthin,  265,  294 
Mett's     method     for     determination      of 

peptic  activity,  17 
Mett's  tubes,  preparation  of,  18 
Micro-organisms    in    urinary    sediments, 

351,  361 
Milk,  218 

citric  acid  in,  218 

detection    of    calcium    phosphate    in, 
224 
lactose  in,  225 
preservatives  in,  225 
difference  between  human  and  cow's, 

219 
experiments   on,    221 
formation  of  film  on,  218,  221 
freezing-point  of,  218 
guaiac  test  on,  222 
influence  of  rennin  on,  128 
isolation  of  fat  from,  225 
Kastle's  peroxidase   reaction   of,  222 
microscopical  appearance  of,  219,  221 
preparation  of  caseinogen  from,  223 
properties    of    caseinogen    of,    223 
quantitative   analysis   of,   410 
reaction  of,  218,  221 
separation  of  coagulable  proteins  of, 

224 
specific  gravity  of,  218,  221 
Millon's  reaction,  90 

reagent,  preparation  of,  91 
Mohr's     method     for     determination     of 

chlorides,  395 
Molisch's  reaction,  23 
Molybdic    solution,    preparation    of,    57 
Monamino   acid  nitrogen,   62 
Monosaccharides,  21,  22 

Barfoed's  test  for,   31,   313 
classification   of,   21 
Moreigne's  reaction   for  uric  acid,   275 

reagent,  preparation  of,  275 
Morner-Sjoqvist-Folin  method  for  deter- 
mination of  urea,  378 
Morner's    reagent,    preparation    of,    84 

test  for  tyrosine,  84 
Motor    and    functional    activities    of    the 

stomach,  128 
Mucic  acid,  36,  40,  337 

test,   36,   41,   337 
Mucin,   56,  87,   89 

biuret  test  on,  56 
hydrolysis  of,   57 
isolation  of,  from  saliva,  56 
Millon's    reaction    on,    56 
Mucins,  87,  89 
Mucoid,    87,    106,    228 
experiments  on,  229 
hydrolysis    of,    229 


Mucoid,  in  urine,   290,  320 

preparation  of,  from  tendon,  228 
Mucoids,  87,  89 
Murexide  test,  274 
Muscle  plasma,  235,  242 

formation  of  myosin  clot  in,  235 
fractional    coagulation    of,    235, 

242 
preparation  of,   241,   242 
reaction  of,   237,  242 
Muscular   tissue,   235 

commercial  extracts  of,  240 
experiments  on  "  dead,"  243 

"  living,"    241 
extractives  of,  236,  241 
fatigue  substances  of,   240 
formulas   of  nitrogenous  extrac- 
tives of,  241 
glycogen   in,   236,    244 
involuntary,  235 
lactic  acid  in,  237,  240,  243 
nonstriated,  235 
pigment   of,   240 

preparation    of    glycogen    from, 
244 
muscle    plasma    from,     241, 
242 
proteins  of,  235 
reaction   of   living,   237 
separation   of   extractives    from, 

245 
striated,   235 
voluntary,  235 
Myohsematin,  240 
Myosan,  87 

formation  of,  243 
Myosin,   235 

biuret  test  on,  243 

coagulation  of,  243 

preparation  of,   243 

solubility  of,  243 
Myosinogen,    235 
Myristic   acid,   218 
Myristin,  133 
Myrtle  wax   (see  Bayberry  tallow,   136) 

Nakayama's    reaction   for   bile   pigments, 

i55,    324 
reagent,  preparation  of,   155,  324 
Nencki    and    Sieber's    reaction    for    uro- 

rosein,  341 
Neosine,  236 

formula  for,  241 
Nervous  tissue,  248 

constituents  of,  248 
experiments  on  lipoids  of,  250 
lipoids   of,   248,    250 
percentage  of  water  in,  248 
phosphorized    fats    of,    248 
proteins   of,   248 
Neurokeratin,    248 

Neutral   olive    oil,    preparation   of,    136 
Neutral  sulphur  compounds,  264,  285 
Nitrates   in  urine,   265,   304 
Nitrites  in  saliva,  test  for,  57 
Nitrogen,  62 


[NDEX. 


Nitrogen,    forms    of    ill    protein    molt  i 
62 
importance  of,  in   sustaining   life,  62 
in   urine,  quantitative  determination 

of,  381 
Nitrogen   iodide,   formal  inn    of,   328 
Nitrogenous   extractives   of   muscular    ti- 
sue,  236 
formulas    for,   -'i" 
Nitroso-indole    nitrate    test,    169 
Nitrosothymol,    formation    of    in    Heller's 

test,  315 
Non-nitrogenous  extractives  of  muscular 

tissue,    236 
Normal  urine,  254 

characteristics  of,  254 
constituents  of,   264 
experiments    on,    264-304 
Novaine,   236 

formation  for,  241 
Nubecula,  290,   320 
Nucleic  acid,  87,   107 
Nucleins,    107,   122,  248 
Nucleohistone,  87,  89 
Nucleoproteins,  87,  89,   106,  248,  264 
in  bile,   154 
in  feces,  180 
in  nervous  tissue,  248 
in  urine,  28,  264,  290,  305,-  320 

test  for,  321 
occurrence   of,    107 
Ott's   precipitation   test   for,    321 
Nylander's    reagent,    preparation    of,    30, 
312 
test,  30,  312 

Obermayer's  test  for  indican,  281 

reagent,  preparation  of,  281 
Oblitine,  236 
"Occult"  blood  in  feces,  17s,  178 

tests   for,    178 
Olein,  132 
Olive  oil,   135 

emulsification    of,    136 
neutral,  preparation  of,   136 
Opalisin  in  milk,  221 
Orcin   test,   38 
Organic     physiological      constituents     of 

urine,  264 
Organized   ferments,    1 
Organized   urinary   sediments,    351 
Osborne-Folin   method  for  determination 

of   total    sulphur   in   urine,    386 
Ossein,   233 

preparation  of,  233 
Osseoalbumoid.  233 
Osseomucoid.   87,    106,    233 

chemical  composition  of,  106 
Osseous  tissue,  233 

experiment  on,  233 
Ott's   precipitation   test    for   detection    of 

nucleoprotein  in  urine.  321 
Ovalbumin,  86 
Ovoglobulin,  86 

Oxalated  plasma,   preparation   of.   201 
Oxalic  acid,  264,  284,  408 


1  Ixalic  a<  id,  formula  foi 
in  urine 

quantitative     determinate   1 
408 
1  Ixaluria,  285 
1  >xaluric  at  id,  36  1.  290 
< Ixamide, 
( Oxidases,  .-.•  1 
Oxyacids,  [62,  1  '•;,  171 

tests  for,   171 
,;  oxj  but)  rir  acid, 

Black's    method    foi    determina- 
tion of,    1 "  ', 
I'.i.m  k's  r<  action  for,  332 
formula    for,    ,i.]2 
Kiilz's  test   for,  .133 
origin  of,   332 
polariscopic  examination  I 
quantitative     determination     of, 

402 
Shaffer's  method  for  determina- 
tion of,  402 
Oxyhemoglobin,    62 

Reichert's  method  for  crystallization 

of.    201 

crystalline  forms  of,  186-189 
Oxymandelic  acid,  264,  288 
Oxyproline,    81 
Oxyproteic  acid.   264,  285.  342 

Paduschka-Underhill-Kleiner  method   for 
quantitative  determination  of  allantoin. 

407 

Palmitic   acid.    132,    143 

crystalline  form  of,   137 
experiments  on,   138 
formula    for,    132,    143 
preparation  of,   137 
Palmitin,   132 
Pancreatic  amylase,  141,  142.  14; 

digestion  of  dry  starch  by,    143, 
148 
inulin   by,    14X 
experiments  on,   147 
influence  of  bile  upon  action  of, 
148 
metallic    -alts    upon        1     1 
of.  147 
most   favorable   temperature   for 
action    of,    1  IT 
Pancreatic  digestion,    140 

general  experiments  on.   145 
products  of,   141,   1  »5 
Pancreatic  insufficiency,  Schmidt's  nuclei 

test    for,    1 S 1 
Pancreatic  juice.    [40    143 

artificial,    preparation    of,     I4J 
daily   excretion   of,    M' 
enzymes  of,   141 
freezing  point    of,    >  41 

mechanism    of    secretion    «i.    14° 
reaction    of,    140 
solid   content   of,    1  41 
Specific    e,r.i\  ity   of,    14' 
Pancreatic    lipase.    13a,    14  >•    143 

(  xperiments  on 


440 


INDEX. 


Pancreatic  lipase,  ethyl-butyrate  test  for, 
149 

litmus-milk  test  for,   148 
Pancreatic   protease    (see   Trypsin,   p.    1) 
Pancreatic  rennin,  141,  143 

experiments  on,  149 
Papain,    10 

Para-cresol-sulphuric  acid,  264,  279 
Paradimethylamino     benzaldehyde     solu- 
tion, preparation  of,   170 
Paralactic  acid,  237,  265,  291 
Paramyosinogen,  235 
Paranucleoprotagon,  248,  250 
Paraoxyphenylacetic   acid,    162,    168,   264, 

287 
Paraoxyphenyl-a-amino-propionic         acid, 

Paraoxyphenylpropionic    acid,     162,     168, 

264,  287 
Paraphenelenediamine  hydrochloride,  226 
Parasites,    175,   351.   361 
Paraxanthine,   265,  294 
Parietal  cells,   119 
Parotid   glands,    characteristics    of   saliva 

secreted  by,  53 
Pathological   constituents   of   urine,    305 
Pathological  urine,  254,  305 
constituents    of,    305 
experiments  on,   305-342 
Pektoscope,  260 
Pentapeptides,   88 
Pentoses,  21,  37 

experiments  on,  37 
in  urine,  305,  335 
tests  for,   33s 
Pepsin    (see   Gastric   Protease),   2,   9,    120 
action  of,  influence  of  bile  upon,  128 
influence  of  different  acids  upon, 

85 
metallic  salts  upon,   127 
temperature  upon,   126 
conditions  essential  for  action  of,  120 
differentiation    of,    from    pepsinogen, 

126 
formation  of,    120 
digestive  properties  of,   120 
most  favorable  acidity  for  action  of, 

120 
proteolytic  action  of.  120 
Pepsin-hydrochloric   acid,    125-127 
Pepsinogen,  5,  120,  122 

differentiation  of,   from  pepsin,   126 
formation  of,    120 
extract  of,  preparation  of,  122 
Peptic      activity,      Fuld      and      Levison's 
method   for   determination   of, 
18 
Mett's    method     for    the    deter- 
mination of,  17 
Peptic  proteolysis,   120 

products   of,    120 
relation  of,  to  tryptic  proteolysis, 
121 
Peptides,  64,  66,  88,   114,   115 
Peptone,  63,  88,  89 
ampho,  88,  89 


Peptone,  anti,   88,  89 

differentiation  of,  from  proteoses,  114 
experiments  on,   115 
in  urine,  305,   319 
tests  for,  319 
separation  of,  from  proteoses,   114 
Periodide  test  for  choline,   252 
Peroxidases,  221 

Pettenkoper's  test  for  bile  acids,  156,  325 
Mylius's        modification 

of,  156,  326 
Neukomm's        modifica- 
tion of,   156,   326 
Phenaceturic  acid,  265,  291 
Phenol,   162 

tests  for,    170 
Phenolphthalein  as  indicator,   124 

preparation  of,  124 
Phenol-sulphuric  acid,  264.  279 
Phenyl-a-amino    propionic    acid,    69 
Phenylalanine,    63,   69 
Phenyldextrosazone,  24 

crystalline    form    of,    Plate    III,    op- 
posite p.  24 
Phenylhydrazine.   24,   25 

acetate  solution,  preparation  of,  24 
mixture,  preparation  of,  24 
reaction,    24 

Cipollina's  modification  of,  25 
Phenyllactasazone,     crystalline    form    of, 

Plate    ill,    opposite   p.   24 
Phenylmaltosazone    crystalline    form    of, 

Plate  III,  opposite  p.  24 
Phenylpotassium   sulphate,   279 
Phosphates  in  urine,  265,  299 
detection  of,   301 
experiments  on,   301 
quantitative      determination     of, 

389 
Phosphatides,    87,    151 
Phosphocarnic  acid,  236,  241,  265,  292 
Phosphoproteins,   87,   88,   107 
Phosphorized    compounds    in    urine,    265, 

291 
Physiological  constituents  of  urine.   264 
Pigments    of   urine,    254,    265,    292 
Pine  wood  test  for  indole,  169 
Piria's  test  for  tyrosine,   83 
Polariscope,  use  of,  32 

in  detection  of  conjugate  glycur- 

onates,   334 
in  determination  of  dextrose,  32 
/3-oxybutyric  acid,   333 
Polypeptides,   64,   66 
Polysaccharides,  22,  43 
classification  of,  22 
properties  of,  43 
Posner's  modification  of  biuret  test,  93 
Potassium  in  urine,  265,   302 
Potassium  indoxyl-sulphate   (see  Indican, 
pp.    162,   280) 
formula   for,    163,   280 
origin   of,    162,    179 
tests  for,  280 
Primary  protein   derivatives,   87 
Primary  proteoses,    114 


[NDEX. 


44  ' 


Products  of  protein  hydrolysis,  63,  <>; 

Prolamins,    105 

classification  of,  87,  88 
Proline,  63,  80,    105,    141 

crystalline   form   of   laevo-o-,  80 
crystalline  form  of  copper  salt  of,  Bi 
Prosecretin,    140 
Protagon,  248,  249 

preparation  of,  250 
Protamines,  classification  of,  86 
Proteans,   87,    108 
Protease,  gastric,  9 

experiments  on,  9 
pancreatic,  9 

experiments  on,  9 
vegetable,   10 
Proteases,   9 

experiments  on,  9 
Proteins,   61 

acetic     acid     and     potassium     ferro- 

cyanide  test  for,  99 
Acree-Rosenheirn   test  on,  93 
action   of  alkaloidal   reagents   on,   97 
action  of  metallic  salts  on,  97 

mineral    acids,    alkalies    and    or- 
ganic acids  on,  96 
Adamkiewicz  reaction  on,  91 
Bardach's  reaction  on,  94 
biuret  test  on,  91 
chart  for  use   in  review   of,    117 
chemical  composition  of,  61 
classification  of,  85,  86,  88 
coagulation  or  boiling  test  for,  99 
color  reactions  of,  90 
conjugated,  87,  89,   106 
decomposition  of,  62 
by   hydrolysis,    63 
by   oxidation,   6^ 
products   of,   63 

experiments  on,  82 
separation  of,  82 
study  of,  63,  82 
derived,    87 

formation   of  fat  from,    134 
formulas  of,   62 
Heller's  ring  test  on,  97 
importance    of,   to   life,    61 
Hopkins-Cole  reaction  on,  91 
in  urine,   305,   313 
test   for,    314 
Liebermann's   reaction   on,    93 
Millon's   reaction  on,   90 
molecular   weights   of,   62 
Posner's  reaction  on,  93 
precipitation  of,  by  alcohol,    100 
alkaloidal  reagents,  97 
metallic  salts,  97 
mineral   acids,   96 
precipitation  reactions  of.  95 
quantitative     determination      of,      in 

milk,  412 
review   of.    11 5 
Robert's  ring  test    on,  97 
salting-out  experiments   on,   99 
scheme  for  separation  of,   116 
simple,    86,   89 


Proteins,  Bynthi 

xanthoproteii    rea<  tion  on,  91 
Protein  .  1  oagulated,  1 1 1 

biuret  11.' 

foi  mation  of,   1 1 1 
I  lopkint  <  ole   rea<  tion  on,    1 1 2 
Millon's  reai  tion  on,   1 1 2 
solubility  of,   in,  iu 
xanthoproti  in    on,    112 

I  '1  otein  coagulated   • 
Proteins    1  onjugated,  87, 
classes  of,  87,  106 
experiment     on,    1 95,    196 

201,  203,  -'-".! 
nomenclature  of,  87 
0)  1  urrence  of,    106 
Protein  cystine,    73 
Protein  derivatives,  primarj    I 

secondary,  63,  1 1 3 
Proteins  of  milk,  218,  220,  221 

quantitative     determination     of, 
412 
Proteolytic  enzj 
Proteolysis,  peptic,    t20 

tryptic,    ij  1 
Proteose,  63,  88,  89,   113 

v.   Aldor's  method   for  detection  of, 
320 

biuret    tCst   on,    i  15 

gulation   test   on,   1 1 5 
deutero,  88,  89.   1  1 3 
differentiation  of,  from  peptone,  114 
experiments  on.    1  1  5 
hetero,   89,    113 
in  urine,  305,  313,  319 

test  for,  319 
potassium     ferrocyanide    and    acetic 

test  on.  l  1  s 
powder,  preparation  of,  1 14 
precipitation    of.    by    nitric    acid,    115 

by  picric  acid.    1  1  5 

by  potassio  mercuric  iodide,  115 

by  trichloracetic  acid,   1 15 
primary.    1  14 
proto,  88,  89.    1  1  3 
Schulte's    method    for    detection    of, 

319 
secondary.    1  14 

separation  of,   from   peptones,    114 
Protoproteose.   88,    89 
Proteoses  and  peptones,  88,  89,  113 
separation   of,    114 

tests    on,    114 

Proteose-peptone.   114 
Proteose-peptone,  coagulation  test  on,  114 
experiments  on,  1 1 4 
Millon's  reaction  on,   1  1  { 
precipitation  of,  by  nitric  acid.   114 
by  picric  acid.   1 14 
Prothrombin.   191,   192 
Pseudo  globulin,  182,  183, 
Ptomaines  and  leucomaines  in  urine 

294 
Ptyalin    (see  Salivarj    amj 
Purdy's  method  t'..r  determination    if  dea 
trose 


442 


INDEX. 


Purdy's  solution,  preparation  of,   370 
Purine  bases,  107,  405 

in    urine,    quantitative    determination 

of,  405 

Pus  casts  in  urinary  sediments,   351,  359 

Pus  cells  in  urinary  sediments,   351,   353 

Putrefaction,    indican    as    an    index    of, 

162,  279 
Putrefaction    mixture,    preparation    of   a, 

164 
Putrefaction  products,  162 

experiments  on,    164 
most  important,   162 
tests  for,  168 
Pyloric   glands,    118 
Pyrocatechin-sulphuric  acid,  264,  279 
cc-pyrrolidine-carboxylic   acid   (see   Prolin, 
p.  80) 

Qualitative    analysis    of    the    products    of 
salivary  digestion,   60 
stomach  contents,    129 

Quantitative  analysis  of  blood,  415 
of  gastric  juice,  413 
of  milk,  410 
of  urine,   366 

Quantitative    determination    of    ammonia 
in  urine,  380 
amylolytic  activity,   16 
acetone  in  urine,  400 
acetone     and     diacetic     acid     in 

urine,  397 
acidity  of  urine,  404 
allantom  in  urine,  407 
ash  of  milk,  411 
caseinogen   of  milk,   412 
chlorides  in  urine,  394 
creatine   in    urine,    393 
creatinine,   392 
dextrose  in  urine,  367 
diacetic  acid  in  urine,  401 
fat  in  milk,  410 
hippuric  acid  in  urine,  383 
indican  in  urine,  393 
lactalbumin    in    milk,    412 
lactose  in  milk,  412 
nitrogen  in  urine,  381 
oxalic  acid  in  urine,  408 
/3-oxybutyric   acid   in   urine,    402 
peptic  activity,   17 
phosphorus   in  urine,   389 
protein  in  milk,  412 
protein  in  urine,  366 
purine  bases  in  urine,   405 
sulphur  in  urine,  384 
total  solids  in  milk,  411 
total   solids  in  urine,   408 
tryptic  activity,  19 
urea  in  urine,  374 
uric  acid  in  urine,  372 

Quevenne    lactometer,    determination    of 
specific  gravity  of  milk  by,  410 

Raffmose,  22,  43 
Rancid  fat,  133 
Reaction  of  the  urine,  256,   300 


Reduced  alkali-haematin,  206 

Reduced  haemoglobin,  203 

Reductases,  221 

Reichert's    method    for   crystallization    of 

oxyhemoglobin,  201 
Remont's    method    for   detection    of    sali- 
cylic acid  and  salicylates,  226 
Rennin,  gastric,   121 

action  of,  upon  caseinogen,   121 

experiments   on,    128,    130 

influence  of,  upon  milk,  121,  128 

in   gastric  juice,   absence   of,    121 

nature  of  action  of,  121 

occurrence  of,    121 
Rennin,  pancreatic,  141,  143 
experiments  on,  149 
Reticulin,  106 

Reversibility   of    enzyme    action,    6,    55 
Reynolds-Gunning   test   for   acetone,    329 
Rhamnose,  21,  38 
Ricin,    10,    196 
Ring  test  for  urobilin,  294 
Roaf's   method   for   crystallizing  hippuric 

acid,    283 
Robin's  reaction  for  urorosein,   341 
Robert's  ring  test  for  protein,  97,   315 

reagent,  preparation  of,  97,  315 
Rosenheim's  bismuth  test  for  choline,  253 
Rosenheim's    periodide    test    for    choline, 

252 
Rossi's  reaction  for  indican,  281 
Rubner's  test  for  lactose  in  urine,  337 

Saccharide  group,   22 
Saccharose    (see    Sucrose) 
Sahli's  desmoid   reaction,    127 
Saliva,  53 

alkalinity  of,  54 

amount  of,  54 

bacteria   in,    55 

biuret  test  on,  56 

calcium  in,  54 

chlorides  in,  57 

constituents  of,  54 

digestion  of  dry  starch  by,  58 

digestion  of  inulin  by,  58 

digestion  of  starch  paste  by,  55,  58 

enzymes  contained  in,  54,  55 

excretion  of  potassium  iodide  in,  60 

inorganic  matter  in,  tests  for,  57 

Millon's  reaction  on,  56 

mucin  from,  preparation  of,  56 

nitrites  in,  test  for,  57 

phosphates  in,  test  for,  57 

potassium   thiocyanate   in,   54 

reaction  of,   54,   56 

secretion  of,  53 

specific  gravity  of,  54,  56 

sulphates  in,  test  for,  57 

thiocyanates  in,   54 

tests  for,  57 
Salivary  amylase,    1,   54,    119 

activity  of,   in   stomach,   55,    119 
inhibition  of  activity  of,  55 
nature  of  action  of,  54 
products  of  action  of,  55 


[NDEX. 


Salivary   digestion,   53 

influence    of    acids    and    alkalis 
on,  55.  59 

dilution  on,   59 
metallic    salts    on,    so 
temperature  on,   58 
nature    of    action    of    acids    and 

alkalis   on,   59 
qualitative   analysis   of   products 
of,  60 
Salivary  digestion  in  stomach,  55,   119 
Salivary    glands,    53 
Salivary   stimuli,   53 

Salkowski-Autenrieth-Barth     method     for 
determination   of   oxalic   acid   in    urine, 
408 
Salkowski's  method  for  determination  of 

purine  bases,  407 
Salkowski-Schippers     reaction      for     bile 

pigments,    155.   325 
Salkowski's  test  for  cholesterol,   158,  252 

for  creatinine,  278 
Salmine,  87,  88 

Salted  plasma,  preparation  of,  201 
Salting-out   experiments   on   proteins,    95, 

99 
Sarcolactic  acid,   237 
Scallops,    preparation    of    glycogen    from, 

244  . 

Schalfijew's    method    for    preparation    of 

hsemin,   199 
Scheme  for  analysis  of  biliary  calculi,  158 
bone  ash,  234 
stomach  contents,  130 
urinary  calculi,  364 
separation  of  carbohydrates,  51 
of   proteins,    116 
Scherer's    coagulation    method    for   deter- 
mination of  albumin  in  urine,  366 
Schiff's  reaction  for  cholesterol,   159,  252 

for  uric  acid,  275 
Schiff's  reagent,  preparation  of,   159,  252 
Schmidt's    nuclei   test    for   pancreatic    in- 
sufficiency,   181 
Schmidt's  test  for  hydrobilirubin,   179 
Schulte's    method    for    detection    of    pro- 
teose in  urine,   319 
Schumm's  modification  of  the  guaiac  test, 

196 
Schiitz's   law,  statement  of,   7,    18 
Schweitzer's    reagent,    action    of,   on    cel- 
lulose, 50 
preparation  of,  50 
Scleroproteins,    85    (see    Albuminoids) 
Scombrine.   87 
Scombrone,  86,  88 
Secondary  protein  derivatives,  88 
Secondary   proteoses,    114 
Secretin,    140 
Seliwanoff's  reaction,   35,   339 

reagent,  preparation  of,   35,   339 

Separation    ot    feces,    importance    of.    in 

nutrition   and  metabolism   experiments, 

174 
Serine,   63.   69 

crystalline   form   of,   69 


Serine,   formula    for, 
Serum  albumin,  81 

in    .11111.  . 

Serum  globulin, 

in    111 111.  .     |OS,   318 
test    t'.r,    318 
Shaffer's    method    for    determination    of 

ii  oxybutj  rii    a.  id, 
Sherman's    compressed  1  -hod 

fur  determination  ulphur  in 

ur  in. 
Sherrington's    solution,    preparation 

- '  •' 
Silicates  in  urine,  265,  .',.,  1 

SI  al..l. !,    [62,    1  70 

for,    1  ;.. 
Skatole-carbonic   acid,    167 

test   for,    171 
Smith's  test  for  bile  pigments,  155,  325 
Soap,  salting-out    of,    137 

Soaponilii  atioii,    132 

of   lard,    138 
Sodium  and  potassium  in  urine.  J65,  302 
Sodium    alizarin    sulphonate   as   indii 
125 
preparation    of,    125 
Sodium    chloride,   crystalline    form.    200 
Sodium   chloride   in   urine,   265,   298,   394 
Sodium  hydroxide  and  potassium  nitrate 
fusion     method     for    determination     of 
total   sulphur  and   phosphorus   in   urine, 
3&7.  390 
Sodium     hypobromite    solution,    prepara 

tion  of,  375 
Sodium  sulphide  solution,  preparation  of, 

406 
Solera's    reaction    for    detection    of   thio- 
cyanate  in   saliva,   57 
test    paper,    preparation    of,    57 
Soluble  starch,   8,   54 
Soxhlet   apparatus   for   extraction   of   fat. 

41  o 
Soxhlet      lactometer,      determination      of 

specific   gravity  of   milk   by,    410 
Specificity  of  enzyme  action,  6 
Spectroscope,     use    of,     in     detection    of 

blood,   323 
Spermatozoa    in    urinary    sediments,    351, 
360 
microscopical   appearance   of   human. 
360 
Spiegler's   rim;   tesl    for  protein,  98,  316 

reagent,  preparation   of,   98,  3'6 
Sprigg's    method     for    determination    of 

peptic  activity,    17 
Standard     ammonium     thiocyanate     solu- 
tion,  preparation   ol 

ntic    nitrate    solution,    prepara 
tion  of,    ;w> 
uranium  lution,    prepara 

tion  of,  389 
Starch.   22,   4.? 

action  of  alcohol  on  iodide  ol 

action   of   alkali   on   iodide   ol 
heat   on   iodide  of,    I" 


444 


INDEX. 


Starch,    dry,    digestion    of,    by   pancreatic 
amylase,    143,    148 
dry,    digestion    of,    by    salivary    amy- 
lase,   58 
experiments  on,  44 
iodine  test  for,  44 
microscopical   characteristics   of,   43 
microscopical   examination   of,   44 
potato,  preparation  of,  44 
soluble,    54 
solubility    of,    44 
various  forms  of,  45 
Starch   group,   22 

Starch  paste,  action  of  tannic  acid  on,  46 
diffusibility   of,    46 
digestion  of,  by  pancreatic  amy- 
lase,   142,    147 
by   salivary  amylase,   54,   58 
Fehling's  test  on,  46 
hydrolysis  of,  46 
iodic  acid  paper,  58 
preparation  of,  44 
Steapsin    (see   Pancreatic  lipase,    132) 
Stearic  acid,  249 
Stearin,  133 

Stellar  phosphate,  224,  345 
Stercobilin,    173 
Stokes'  reagent,  action  of,  203,  206 

preparation  of,  203 
Stomach,  motor  and  functional  activities 

of,  128 
Stomach    contents,    lactic    acid    in    tests 
for,    129 
qualitative   analysis   of,    129 
Stone-cystine,    73 
Sturine,  87 
Sublingual      glands,      characteristics      of 

saliva  secreted  by,  53 
Submaxillary    glands,     characteristics     of 

saliva  secreted  by,  53 
Substrate,  2,  6 
Sucrase,   11,   144 

experiments  on,  11 
vegetable,    1 1 
Sucrose,   21,   41 

experiments  on,  42 
inversion  of,  41 
production  of  alcohol  from,  42 
structure   of,   42 
Sulphanilic    acid,    342 
Sulphates  in  saliva,  test  for,  57 
Sulphates  in  urine,  265,   296 
experiments  on,  297 
ethereal,    279,    296 

quantitative      determination 
of,  386 
inorganic,  296 

quantitative      determination 
of,  385    _   . 
total,  quantitative  determination 
of,  384 
Sulphocyanides   (see  Thiocyanates,  54) 
Sulphur  in  protein,   102 

loosely  combined,  test  for,   102 
in   urine,    quantitative    determination 
of,    384 


Sulphur  in  acid,   102 

lead  blackening,    102 

mercaptan,    102 

neutral,   296 

oxidized,    102 

unoxidized,    102 
Suspension  of  manganese  dioxide,   406 

Tallow  bayberry,  saponification  of,  136 
Tallquist's   haemoglobin   scale,   determina- 
tion of  haemoglobin  by,  212 
Tannic  acid,  influence  of,  on  dextrin,  49 

on   starch,   46 
Tannin  test  for  carbon  monoxide  haemo- 
globin, 205 
Tanret's  reagent,  preparation  of,  98,   317 
Tanret's  test,  98 
Tartar,  formation  of,  54 
Taurine,    151,  240 
derivatives,   264 
formula   for,    151,   240 
preparation  of,   159 
Taurocholic   acid,    151 

group,    151 
Taylor's   test   for   acetone,    329 
Teichmann's  crystals,  form  of  (see   Haemin 

crystals,  p.  198) 
Tendomucoid,  87,  106,  228 
biuret  test  on,  229 
chemical   composition   of,    106 
hydrolysis  of,  229 
loosely  combined  sulphur  in,  test  for, 

229 
preparation  of,  228 
solubility  of,  229 
Tetrapeptides,  88,  89 
Thiocyanates    in    saliva,    significance    of, 

54 
ferric   chloride   test   for,    57 
Solera's   reaction   for,   57 
Thiocyanates  in  urine,  264,  285 
Thiophene,    129 

Thoma-Zeiss   haemocytometer,    212 
Thrombin,    191,    192 
Thymus  histone,   86 
Thymol,    formula   for,    262 

interference   of,   in   Lieben's   acetone 

test,  329 
interference  in  Heller's  ring  test,  315 
use  of,  as  preservative,  262 
Tincture  of  iodine,   preparation   of,  424 
Tissue,  adipose,  experiments  on,   131,  234 
connective,    2.2"] 

white  fibrous,  227 

composition  of,   228 
experiments  on,  228 
yellow   elastic,   230 

composition     of,     230 
experiments  on,  231 
epithelial,    227 

experiments  on,  227 
muscular,   235 

experiments    on,    241 
nervous,    248 

experiments    on,    250 
osseous,  233 


INDEX. 


Tissue,  osseous,  experiment  on,  233 
Tissue   debris  in   urinary   sediments,   351, 

361  . 
Toison's   solution,   preparation   of,    21  1 

Tollen's    reaction    011    conjugate    glycuro 

nates,  33  1 
galactose,  36 
arabinose,   37 
Topfer's  method  for  quantitative  analysis 

hi  gastric  juice,  41 3 
Topfer's  reagent,  as  indicator,   i_>3 

preparation  of,   123 
Total  solids,  of  milk,  quantitative  deter 
initiation  of,  41 1 
of  urine,  quantitative  determina 
tion  of,  408 
Total    sulphur   of   urine,   quantitative   de- 
termination of,  386-389 
phosphorus     of     urine,     quantitative 
determination  of,  390 
Tri-butyrin,  218 
Trimethyl-oxyethyl-ammonium   hydroxide 

(see  Choline,  248) 
Tri-olein,  133,  218 
Tri-palmitin,   132,  143,  218 
Tri-stearin,   133,  218 

Trichloracetic   acid,   precipitation    of   pro- 
tein by,  97 
Trioses.  22 
Tripeptides,   88,   89 
Triple  phosphate,  247,  301,   344 
crystalline    form    of,    301 
formation  of,  301 
Trisaccharides,  2.2,  43 
Trommer's  test,  z"j 
Tropaeolin   00,   as   indicator.    124 

preparation  of,   124 
Trypsin     (see    also     Pancreatic    protease, 
1  1   ■» 
action    of,   upon    proteins,    64 
experiments  on,    145 
influence  of  alkalis  and  mineral  acids 

upon,   141 
nature  of,   141 
pure,  preparation  of,  141 
Trypsinogen,  5,   141 

activation   of.    142 
Tryptic  digestion,   140 

influence  of  bile  on,   146 

metallic  salts  on,   146 
most  favorable  reaction  for,  145 

temperature   for,    146 
products  of,    14'.    T45 
Tryptic  proteolysis,    121,    141 
Tryptophane,  63,  73,   141,   145 
bromine  water  test  for,  145 
formula   for.   73 

group  in  the  protein  molecule.  91 
Hopkins-Cole  reaction   for.   91 
occurrence    of,    as    a    decomposition 

product    of  protein,   63,    73 
occurrence  of.  as  an  end-product  of 
pancreatic  digestion,  141.   1 451 
"Twinning"  of  oxyhemoglobin   crystals, 

190 
Tyrosine,   63.   70,   90,    141 


1 

1  imenti  on,  x.\ 
formula  1 

l  [offmann'i  rea<  tion      1 
in  urinary  sedii 
mil  roscopii  d    •  ■•  amination    oi 
Morner'i   t>  si   1 

■  ■I  1  uri  iip  • 

Piria'a  test  tor,  83 

sails    of,    71 

separation  of,  from 
solubility  ol 
sublimation  of,  x.i 
Tyrosine  sulphui  ii 

\ .  I  Idranskj  's  test  for  bile  a<  id 
rficlni.mn's  reagent,  preparation  ol 

reaction  for  lactic  acid,  1  .•■; 
Unknown  substances  in  urine, 
Unorganized   ferments,   1 
Uranium   acetate   method    for   detei 

tion  of  total  phosphates  in  urine,  389 
Uracil,    107 

Wheeler-Johnson   reaction    for, 
I 'rate,    ammonium,    crystalline    form    of, 
Plate    VI,   opposite   p.    348 
sodium,    crystalline    form    of,    348 
Urates  in  urinary  sediments,  344. 
Urea,  264,  265 

crystalline  form  of,  266 
decomposition    of,    by    sodium    hyp" 

bromite,   268,   271 
excretion   of,   266 
experiments  on.  269 
formation  of,  267 
formula   for,   265 
furfurol    test    for,    271 
isolation  of,  from  the  urine.  269 
melting-point  of,  269 
quantitative  determination  of,   374 
Urea  nitrate,   270 

crystalline    form    of,    268 
formula  for,  268 
oxalate,  270 

crystalline  form  of,  270 
formula  for,  268 
Urethral    filaments   in   urinary   sediments. 

35*1   360 
Uric  acid.   28,   236,   241,   264,   271 
stalline  form  of  pun 
endogenous 

■    Molls,      27a 

experiments  on, 

formula  for.  271 
in  leukaemia,  273 
in    urinary    sedimi  I 

crystalline   torm  of    Plate   V, 

opposite  ; 

"on   of,   from   the  urir 

Moreigne's    n  action    for, 
murexide  test  t'..r. 
origin   of, 
quantitative     determinati 

3T- 

Folin  Schaffer     mi 
for, 


446 


INDEX. 


Uric  acid,   quantitative   determination   of, 
Heintz  method  for,  373 
Kriiger    and    Schmidt's 
method  for,  373 
reducing  power  of,  28 
Schiff's  reaction  for,  275 
Uricolytic  enzymes,  3,  14 

experiments  on,  14 
Urinary  calculi,  362 

calcium  carbonate  in,  363 

oxalate  in,   363 
cholesterol  in,   365 
compound,    362 
cystine  in,  363 
fibrin  in,  363 
indigo  in,  365 
phosphates  in,  363 
scheme  for  chemical  analysis  of, 

364 
simple,   362 

uric  acid  and  urates  in,  363 
urostealiths  in,  363 
xanthine  in,  363 
Urinary   concrements  (see  Urinary  calculi, 

p.   362) 
Urinary  concretion    (see   Urinary   calculi, 

p.  362) 
Urination,  frequency  of,  256 
Urinary  sediments,  343 

ammonium      magnesium      phos- 
phate in,  344 
animal  parasites  in,  351,   361 
calcium   carbonate   in,    345 
oxalate    in,    344 
phosphate  in,  345 
sulphate  in,  346 
casts  in,   351,  354 
cholesterol  in,   349 
collection  of,  343 
cylindroids   in,   359 
cystine   in,    348 
epithelial  cells  in,  351,  352 
erythrocytes  in,  351,  360 
fibrin    in,    351,    361 
foreign    substances   in,    351,    361 
hffimatoidin     and     bilirubin     in, 

344,350  _ 
hippuric  acid  in,   349 
indigo  in,   344,   351 
leucine  and  tyrosine  in,  344,  349 
magnesium    phosphate    in,     344, 

35o_ 
melanin  in,   344,   351 
micro-organisms    in,    351,    361 
organized,   343 
pus  cells  in,  351,  353 
spermatozoa  in,  351,  360 
tissue  debris  in,  351,  361 
unorganized,  343,  351 
urates   in,    344,    347 
urethral   filaments  in,    351,    360 
uric  acid  in,  346 
xanthine  in,  344,  350 
Urine,  254-409 

acetone  in,  327 
acidity  of,  256,  300 


Urine,  acid  fermentation  of,  258 
albumin  in,   305,   314 
alkaline   fermentation   of,   256 
alantoin  in,   264,   285 
ammonia  in,  257,  265,  295 
aromatic  oxyacids  in,  264,  287 
benzoic   acid  in,   264,  289 
bile  in,   324,   325 
blood  in,  305,  321 
calcium  in,  303 
carbonates  in,  265,  303 
chlorides  in,  265,  298 
collection  of,  262 

conjugate  glycuronates  in,   305,   334 
color  of,   254 
creatinine   in,   264,   275 
dextrose  in,   305 
diacetic  acid  in,  305,  331 
electrical    conductivity    of,   261 
enzymes  in,  265,   290 
ethereal    sulphuric   acid   in,    264,    279 
fat  in,   305,  335 
fluorides,  in,  265,   298,   394 
freezing-point  of,  260 
galactose  in,   305,   337 
general  characteristics  of,  254 
globulin  in,  305,  318 
Haser's  coefficient  for  solids  in,  259, 

409 
hsematoporphyrin  in,  305,  336 
hippuric   acid   in,   282,   349 
hydrogen  peroxide  in,  265,  304 
inorganic    physiological    constituents 

of,   295 
inosite    in,    305,    339 
iron  in,  265,  303 
lactose  in,  305,  336 
lsevulose  in,  305,  338 
laiose   in,    305,    340 
leucomaines   in,    265,    294 
Long's  coefficient  for  solids  in,  259, 

409 
magnesium   in,   265,   303 
melanin  in,  305,  340 
neutral  sulphur  compounds  in,  285 
nitrates   in,   265,    304 
nucleoprotein   in,   264,   290,   305,   320 
odor  of,  256 
organic  physiological  constituents  of, 

264 
oxalic  acid  in,  264,  284 
oxaluric  acid  in,  264,  290 
/3-oxybutyric  acid  in,  332,  402 
pathological  constituents  of,  305 
paralactic  acid  in,  237,  265,  291 
pentoses  in,   305,   335 
peptone   in,   305,    319 
phenaceturic  acid  in,  265,  291 
phosphates  in,  265,  299 
phosphorized  compounds  in,  265,  291 
physiological    constituents    of,    264 
pigments  of,  254,  265,  292 
potassium  in,  265,  302 
proteins  in,   305,   313 
proteoses  in,   305,  313,   319 
ptomaines  in,  265,  294 


[NDEX. 


Urine,  purine  bases   in,    jos 

quantitative  analysis  (if,  366 

reaction   of,  256,  300 

silicates  in,  265,  304 

sodium  in,  265,  302 

solids  of,  259,  408 

specific  gravity  of,  258 

sulphates  in,  265,  296 

transparency  of,  255 

unknown    substances    in,    305,    342 

urea  in,  264 

uric  acid  in,  264,  271 

urorosein    in,    305,    341 

volatile  fatty   acids   in,   265,   290 

volume    of,    254 
Urobilin,  254,  265,  292 

tests  for,  293 
Urochrome,  254,  292 
Uroerythrin,   10,  254,  292,  312 
Uroferric  acid,  264,  285,  342 
Uroleucic  acid,  264,  288 
Urorosein,  305,  341 

tests  for,  341 

Valine,    75 
Vegetable  amylase,  9 

lipase,    10 

protease,  9 

sucrase,  11 
Vegetable  globulins,  86 
Vegetable  gums,  22 
Veith       lactometer,       determination       of 

specific   gravity   of  milk  by,   410 
Viscosity  test,  57 
Vitellin,   87,  88 

Volatile  fatty  acids,  162,  165,  265,  290 
Volhard-Arnold    method    for    determina- 
tion of  chlorides,  396 
Volume  of  the  urine,  254 

Wax  myrtle,  136 

Waxy    casts    in    urinarv    sediments,    351, 
358 


WYImt's  guaiai  blood   in    ■■ 

17H 
Weinland,  formatioi 

for  en  atinine,  278 
Wheeler  [ohnson  reaction  for  uracil  and 

to    itn  .     1  ■■; 

White  fibrous  1  onne<  live  ti 

Wirsin  r   urobilin 

Wohlgemuth's    method    for    quantil 
determination  <•(  amylolytic  activity,  \(> 

Xanthine,  241 

crystalline    form    of,    239 

formula  for.  241 

in  urinary  sediment  - 

isolation   of,    from    m.  at    • 

Weidel's  reaction   for.  247 
Xanthine    bases    (see    Purine    bases,    pp. 

107,  40s) 
Xanthine   siher   nitrate,   246 

crystalline    form    of.    247 
Xanthoproteic   reaction,  91 
Xylose,  21,  38 

orcin    reaction    on,    38 

phenylhydrazine    reaction    on,    38 

Tollens'  reaction  on.  37 

Yellow   elastic   connective   tissue 

composition    of,   230 
experiments  on,  231 

Zappert   slide,    216 

Zein,    105 

Zeller's  test   for  melanin,   341 

v.  Zeynek  and  Nencki's  hxmin  test.   199. 

322 
Zikel  pektoscope,   260 
Zymase,  preparation  of,  2 
Zymo-exciter,   5 
Zymogen,    5,    120 


H31 


siological  chemistry 


